U.S. patent application number 12/504600 was filed with the patent office on 2010-02-04 for yeast based expression of proteases and methods of use.
This patent application is currently assigned to Burnham Institute for Medical Research. Invention is credited to Michael Cuddy, Hideki Hayashi, John C. Reed.
Application Number | 20100028892 12/504600 |
Document ID | / |
Family ID | 41608747 |
Filed Date | 2010-02-04 |
United States Patent
Application |
20100028892 |
Kind Code |
A1 |
Reed; John C. ; et
al. |
February 4, 2010 |
YEAST BASED EXPRESSION OF PROTEASES AND METHODS OF USE
Abstract
This disclosure generally relates to components and methods of
using a high throughput screening (HTS) systems for intracellular
proteases, using Caspases as a prototype. Genetic systems are
disclosed for monitoring exogenous caspase activation pathways in
the yeast, Saccharomyces cerevisiae. The yeast-based cellular
systems permit facile expression of proteases (e.g., caspase) and
protease-activating proteins in combinations that reconstitute
entire mammalian pathways in these simple eukaryotes. Among the
assay methods integrated into the yeast system are cleavable
reporter gene activators, in which protease-mediated cleavage
activates a transcription factor. Exemplary systems rely, singly or
in concert, on exogenous recombinant caspases and exogenous
upstream activators of caspases to cleave a chimeric protein giving
rise to a transcription factor which induces the expression of the
LacZ and LEU2 genes. The activities of these genes result in
colored cultures and impart the ability of the yeast to grow in
leucine deficient media. The intensity of the color is measured by
colorimetry and quantified with OD units. The OD units are directly
proportional to the activity of the caspase in the system. The
method of quantification is referred to as the "readout".
Inventors: |
Reed; John C.; (Rancho Santa
Fe, CA) ; Hayashi; Hideki; (Nagasaki, JP) ;
Cuddy; Michael; (Carlsbad, CA) |
Correspondence
Address: |
DLA PIPER LLP (US)
4365 EXECUTIVE DRIVE, SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Assignee: |
Burnham Institute for Medical
Research
La Jolla
CA
|
Family ID: |
41608747 |
Appl. No.: |
12/504600 |
Filed: |
July 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61081363 |
Jul 16, 2008 |
|
|
|
Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12N 15/81 20130101;
C12N 15/1086 20130101; C12N 15/1055 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
GRANT INFORMATION
[0002] This invention was made in part with government support
under Grant No. GM085255 awarded by the NIH. The United States
government may have certain rights in this invention.
Claims
1. A reporter gene system for monitoring protease activity in
living yeast comprising: singly or in concert a exogenous protease
with or without a exogenous activator of said protease; wherein
said exogenous protease cleaves a protein; wherein said protein is
a transcription factor; wherein said transcription factor induces
the expression of one or more reporter genes; wherein the presence
of one or more reporter genes facilitates the ability of said yeast
to grow in a leucine deficient media; wherein a color culture is
generated by the cleavage of a substrate by a gene product; and
wherein said color culture assists with determining the protease
activity of said exogenous protease.
2. The reporter gene system of 1 claim wherein said protease is
caspase.
3. The reporter gene system of claim 2 wherein said protein is a
chimeric protein.
4. The reporter gene system of claim 3 wherein said one or more
reporter genes are LacZ and LEU2.
5. The reporter gene system of claim 4 wherein said one or more
reporter genes is LEU2.
6. The reporter gene system of claim 5 wherein said color culture
is blue.
7. The reporter gene system of claim 6 wherein said blue color
culture is generated by the cleavage of X-gal by the LacZ gene
product, .beta.-galactosidase.
8. The reporter gene system of claim 7 wherein the intensity of
said blue color is measured by colorimetry and quantified with OD
units.
9. The reporter gene system of claim 8 wherein said OD units are
directly proportional to the activity of the caspase in the
system.
10. The reporter gene system of claim 9 wherein said OD units
provide a read out value which may be further analyzed to
qualitatively or quantitatively determine caspase activity in the
system.
11. A reporter gene system for monitoring protease activity in
living yeast comprising: singly or in concert a exogenous
recombinant protease and/or a exogenous upstream activator of said
protease; wherein said recombinant protease cleaves a chimeric
protein; wherein said chimeric protein is a transcription factor;
wherein said transcription factor induces the expression of the
LacZ and LEU2 genes; wherein the presence of said LEU2 gene
facilitates the ability of said yeast to grow in a leucine
deficient media; wherein a blue color culture is generated by the
cleavage of X-gal or other substrates by the LacZ gene product,
.beta.-galactosidase; wherein said blue color culture assists with
determining the protease activity of said exogenous recombinant
protease and/or said exogenous upstream activator of said
protease.
12. The reporter gene system of claim 11 wherein said protease
activity is caspase activity; wherein said exogenous recombinant
protease is a exogenous recombinant caspase and said exogenous
upstream activator of said protease is a exogenous upstream
activator of caspase when present.
13. The reporter gene system of claim 12 wherein; the intensity of
said blue color is measured by colorimetry and quantified with OD
units; wherein said OD units are directly proportional to the
activity of the caspase in the system; wherein said OD units
provide a read out value which may be further analyzed to
qualitatively or quantitatively determine caspase activity in the
system.
14. A method of generating and using a genetic system for
monitoring from exogenous caspase activation pathways in yeast
comprising: singly or in concert a exogenous recombinant caspase
with or without an exogenous upstream activator of caspase; wherein
said recombinant caspase cleaves a chimeric protein; wherein said
chimeric protein is a transcription factor; wherein said
transcription factor induces the expression of the LacZ and LEU2
genes; wherein the presence of said LEU2 gene facilitates the
ability of said yeast to grow in a leucine deficient media; wherein
a color culture is generated by the cleavage of X-gal or other
substrates by the LacZ gene product, .beta.-galactosidase; wherein
the intensity of said blue color is measured by colorimetry and
quantified with OD units; and wherein said OD units are directly
proportional to the activity of the caspase in the system.
15. The method of claim 14 where the yeast is Saccharomyces
cerevisiae.
16. A method of generating and using a genetic system for
monitoring from exogenous caspase activation pathways in yeast
comprising: a single-component system wherein said exogenous
caspase is stably over-expressed and autoactivated in a yeast
strain containing a transmembrane cleavable transcription factor;
wherein said transmembrane cleavable transcription factor is
expressed such that a the majority of the cytosolic domain of said
transmembrane receptor is replaced by a chimeric transcription
factor; wherein said chimeric transcription factor comprises the
DNA-binding domain of LexA and the transactivation domain of B42
and is described as LexA-B42 chimeric transcription factor; wherein
between the Fas and the LexA-B42 domains are one or more
tetrapeptide sequences known to be recognized and cleaved by
various members of the Caspase family; wherein an appropriately
matched set of the appropriate caspase to an appropriate chimera
results in a cleavable transcription factor wherein said
transcription factor induces the expression of a reporter gene;
wherein the presence of a reporter gene facilitates the ability of
said yeast to grow in a leucine deficient media; wherein a color
change is generated by the cleavage of a substrate by the gene
product, wherein the intensity of said color is measured by
colorimetry and quantified with OD units; and wherein said OD units
provide a read out value which may be further analysed to
qualitatively or quantitatively determine caspase activity in the
system.
17. The method of claim 16 wherein said reporter gene LEU 2
facilitates the ability of said yeast to grow in a leucine
deficient media.
18. A method of generating and using a genetic system for
monitoring from exogenous caspase activation pathways in yeast
comprising: a single-component system wherein said exogenous
caspase is stably over-expressed and autoactivated in a yeast
strain containing a transmembrane cleavable transcription factor;
wherein a chimera is expressed such that the majority of the
cytosolic domain of said transmembrane receptor is replaced by a
chimeric transcription factor; wherein said chimeric transcription
factor comprises the DNA-binding domain of LexA and the
transactivation domain of B42 and is described as LexA-B42 chimeric
transcription factor; wherein between the Fas and the LexA-B42
domains are one or more tetrapeptide sequences known to be
recognized and cleaved by various members of the Caspase family;
wherein an appropriately matched set of the appropriate caspase to
the appropriate chimera results in a cleavable transcription factor
wherein said transcription factor induces the expression of the
LacZ and LEU2 genes; wherein the presence of said LEU2 genes
facilitate the ability of said yeast to grow in a leucine deficient
media; wherein a blue color culture is generated by the cleavage of
X-gal by the LacZ gene product, .beta.-galactosidase; wherein the
intensity of said blue color is measured by colorimetry and
quantified with OD units; and wherein said OD units provide a read
out value which may be further analysed to qualitatively or
quantitatively determine caspase activity in the system.
19. A method of using the single component system of claim 16 for
drug screening assays where the activities of Caspase 1, 2, 3, 4,
5, 6, 7, 8, 9, and 10 are measured and compounds that inhibit
Caspase 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 are identified.
20. A method of generating and using a genetic system for
monitoring from exogenous caspase activation pathways in yeast
comprising: a two-component system comprising; an exogenous caspase
which is expressed at a low level to maintain an inactive
pro-caspase form, wherein the level of expression of said exogenous
caspase is engineered to prevent autoactivation; an exogenous
upstream or downstream activator; wherein said transcription factor
is expressed such that a the majority of the cytosolic domain of
said transmembrane receptor is replaced by a chimeric transcription
factor; wherein said chimeric transcription factor comprises the
DNA-binding domain of LexA and the transactivation domain of B42
and is described as LexA-B42 chimeric transcription factor; wherein
between the Fas and the LexA-B42 domains are one or more
tetrapeptide sequences known to be recognized and cleaved by
various members of the Caspase family; wherein an appropriately
matched set of the appropriate caspase to an appropriate chimera
results in a cleavable transcription factor wherein said
transcription factor induces the expression of one or more reporter
genes; wherein the presence one or more receptor genes facilitate
the ability of said yeast to grow in a leucine deficient media;
wherein a color culture comprises a color change generated by the
cleavage of a substrate by the gene product; wherein the intensity
of the color is measured by colorimetry and quantified with OD
units; and wherein said OD units provide a read out value which may
be further analysed to qualitatively or quantitatively determine
caspase activity in the system.
21. A method of generating and using a genetic system for
monitoring from exogenous caspase activation pathways in yeast
comprising: a two-component system comprising; an exogenous caspase
which is expressed at a low level to maintain an inactive
pro-caspase form, wherein the level of expression of said exogenous
caspase is engineered to prevent autoactivation; an exogenous
upstream or downstream activator; wherein the combination of said
exogenous caspase and said exogenous activator containing the
transmembrane receptor, CD95/Fas, chimera; wherein said chimera is
expressed such that a the majority of the cytosolic domain of said
transmembrane receptor is replaced by a chimeric transcription
factor; wherein said chimeric transcription factor comprises the
DNA-binding domain of LexA and the transactivation domain of B42
and is described as LexA-B42 chimeric transcription factor; wherein
between the Fas and the LexA-B42 domains are a plurality of
tetrapeptide sequences known to be recognized and cleaved by
various members of the Caspase family; wherein an appropriately
matched set of the appropriate non-autoactivated exogenous caspase
with an exogenous upstream or downstream activator results in the
production of a transcription factor wherein said transcription
factor induces the expression of the LacZ and LEU2 genes; wherein
the presence of said LacZ and LEU2 genes facilitate the ability of
said yeast to grow in a leucine deficient media; wherein a blue
color culture is generated by the cleavage of X-gal by the LacZ
gene product, .beta.-galactosidase; wherein the intensity of said
blue color is measured by colorimetry and quantified with OD units;
and wherein said OD units provide a read out value which may be
further analysed to qualitatively or quantitatively determine
caspase activity in the system.
22. A method of using the two component system of claim 21 for drug
screening assays where the activities of ASC, RAIDD, FADD, Apaf,
and active caspase-9 are measured and compounds that inhibit the
interaction or activity of ASC with pro-caspase-1, RAIDD with
pro-caspase-2, FADD with pro-caspase-8 or pro-caspase-10, an active
mutant of Apaf with pro-caspase-9, active caspase-9 with
pro-caspase-3 or procaspase-7 are identified.
23. The method of claim 22 where the activity of ASC with
pro-caspase-1, RAIDD with pro-caspase-2, FADD with pro-caspase-8 or
pro-caspase-10, and/or the activity of an active mutant of Apaf
with pro-caspase-22 is identified.
24. The method of claim 22 where the activity of an active
caspase-9 with pro-caspase-3 or procaspase-7 is identified.
25. A method of using a two-component system of claim 20 to
identify known or novel upstream activators of pro-caspase-1,
pro-caspase-2, pro-caspase-3, pro-caspase-4, pro-caspase-5,
pro-caspase-6, pro-caspase-7, pro-caspase-8, pro-caspase-9, and
pro-caspase-10; via the introduction of a cDNA library or other
expression conveyance to screen for biomolecular activators of the
pro-caspases.
26. A method of generating and using a genetic system for
monitoring from exogenous caspase activation pathways in yeast
comprising: a plural-component system comprising; an exogenous
caspase which is expressed at a low level to maintain an inactive
pro-caspase form, wherein the level of expression of said exogenous
caspase is engineered to prevent autoactivation; two or more
exogenous upstream or downstream activators; which individually are
expressed at a low level to maintain an inactive form, wherein the
level of expression of said exogenous activators are engineered to
prevent caspase activation unless all three components are present;
wherein the combination of said exogenous caspase and said
exogenous activator contains a transmembrane cleavable
transcription factor; wherein a chimera is expressed such that the
majority of the cytosolic domain of said transmembrane receptor is
replaced by a chimeric or transcription factor; wherein said
chimeric transcription factor comprises the DNA-binding domain of
LexA and the transactivation domain of B42 and is described as
LexA-B42 chimeric transcription factor; wherein between the Fas and
the LexA-B42 domains are a plurality of tetrapeptide sequences
known to be recognized and cleaved by various members of the
Caspase family; wherein an appropriately matched set of the
appropriate non-autoactivated exogenous caspase with the
appropriate combination of two or more exogenous upstream or
downstream activators results in the production of a transcription
factor; wherein said transcription factor induces the expression of
the LacZ and LEU2 genes; wherein the presence of said LacZ and LEU2
genes facilitate the ability of said yeast to grow in a leucine
deficient media; wherein a blue color culture is generated by the
cleavage of X-gal by the LacZ gene product, .beta.-galactosidase;
wherein the intensity of said blue color is measured by colorimetry
and quantified with OD units; and wherein said OD units provide a
read out value which may be further analysed to qualitatively or
quantitatively determine caspase activity in the system.
27. A method of using a plural component system of claim 26 for
drug screening assays where the activities of FAS, FADD, DR5, ASC,
or NALP1 are measured and compounds that inhibit the activity of
FAS with FADD, DR5 with FADD, FADD with pro-caspase-8 or
pro-caspase-10, ASC with NALP1 or other members of the NOD-like
receptor family, or NALP1 or other members of the NOD-like receptor
family with pro-caspase-1.
28. The method of claim 27 where the activity of FAS with FADD is
identified.
29. The method of claim 27 where the activity of DR5 with FADD is
identified.
30. The method of claim 27 where the activity of FADD with
pro-caspase-8 or pro-caspase-10 is identified.
31. The method of claim 27 where the activity of ASC with NALP1 or
other members of the NOD-like receptor family is identified.
32. The method of claim 27 where the activity of NALP1 with
pro-caspase-1 is identified.
33. A method of using a plural component system of claim 16 to
identify known or novel upstream activators of FADD and NALP1 via
the introduction of a cDNA library or other expression conveyance
to screen for biomolecular activators of FADD or NALP1.
34. A method of using a plural component system of claim 16 to
identify known or novel downstream activators of FAS, DR5, or ASC
via the introduction of a cDNA library or other expression
conveyance to screen for biomolecular activators of FAS, DR5, or
ASC.
35. A high throughput screening assay system for identifying lac-Z
reporter gene activity comprising; measuring .beta.-galactosidase
produced by yeast carrying caspase-cleavable reporter proteins; and
assaying the calorimetric product derived from x-gal substrate in
384 well plates, at an optical density of 620 nm.
36. A reporter gene system for monitoring protease activity in
living yeast comprising: singly or in concert a exogenous
recombinant protease and/or a exogenous upstream activator of said
protease; wherein said recombinant protease and/or exogenous
upstream activator cleave a chimeric protein; wherein said chimeric
protein facilitates a transcription factor; wherein said
transcription factor induces the expression of the LacZ and LEU2
genes; or any other substrate of the LacZ gene, beta-galactosidase,
for quantification.
37. The reporter gene system of claim 36 wherein; the presence of
said LacZ and LEU2 genes facilitate the ability of said yeast to
grow in a leucine deficient media; a blue color culture is
generated by the cleavage of X-gal by the LacZ gene product,
.beta.-galactosidase; and said blue color culture assists with
determining the protease activity of said exogenous recombinant
protease and/or said exogenous upstream activator of said
protease.
38. The reporter gene system of claim 36 wherein said protease
activity is caspase activity; and wherein said exogenous
recombinant protease is caspase; wherein said exogenous recombinant
protease is a exogenous recombinant caspase and said exogenous
upstream activator of said protease is a exogenous upstream
activator of caspase when present.
39. The reporter gene system of claim 38 wherein; the intensity of
said blue color is measured by colorimetry and quantified with OD
units; wherein said OD units are directly proportional to the
activity of the caspase in the system; wherein said OD units
provide a read out value which may be further analysed to
qualitatively or quantitatively determine caspase activity in the
system.
40. A method of using a two-component and/or a plural component
system to identify caspase activation protease networks in yeast,
and to isolate and study an exogenous protease network in a
cellular context.
Description
CROSS REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) of U.S. Ser. No. 61/081,363, filed Jul. 16,
2008, the entire content of which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The disclosure generally relates to components and methods
of using a high throughput screening (HTS) systems for
intracellular proteases, using Caspases as a prototype. Disclosed
are yeast-based cellular systems that permit facile expression of
proteases and protease-activating proteins in combinations that
reconstitute entire mammalian pathways in these simple eukaryotes.
Among the assay methods integrated into the yeast system are
cleavable reporter gene activators, in which protease-mediated
cleavage activates a transcription factor.
[0005] 2. Background Information
[0006] Endopeptidases ("proteases") play critical roles in many
biological processes and are often excellent targets for drug
discovery. Development of high throughput screening (HTS) assays
using purified proteases can be relatively straightforward or it
can be quite challenging, particularly when multi-component systems
are required to achieve protease activation. Also, due to
similarity of the active sites of some groups of proteases,
selectivity of chemical inhibitors can be difficult if not
impossible to achieve, highlighting the need for alternative
screening methods for identifying compounds that target upstream
activators of proteases rather than directly inhibiting the
protease of interest.
[0007] Caspases represent an excellent example of a family of
intracellular endopeptidases for which novel HTS tools are desired.
Caspases are cysteine proteases that are conserved throughout the
animal kingdom. The human genome contains at least 10 genes
encoding Caspases. These proteases often collaborate in complex
proteolytic networks that encompass upstream initiators and
downstream effectors, where upstream members of the Caspase family
cleave and activate downstream members. Upstream initiator Caspases
become activated through protein interactions involving assembly of
multi-protein complexes that are difficult to reconstitute in
vitro. The substrates cleaved by active Caspases are responsible
for either apoptotic cell death or for cytokine-mediated
inflammation, thus making these proteases attractive targets for
drug discovery for a wide variety of degenerative diseases,
ischemic disease, autoimmunity, inflammatory conditions, and some
host-pathogen interactions.
[0008] Currently, there is no assay technology for high-throughput
screening which reconstitutes the use and economy as those
facilitated by the present disclosure.
SUMMARY OF THE INVENTION
[0009] Herein are disclosed genetic systems for monitoring
exogenous caspase activation pathways in the yeast, Saccharomyces
cerevisiae. These systems rely, singly or in concert, on exogenous
recombinant caspases and exogenous upstream activators of caspases
to cleave a chimeric protein giving rise to a transcription factor
which induces the expression of the LacZ and LEU2 genes. The
activities of these genes result in blue cultures and impart the
ability of the yeast to grow in leucine deficient media. The blue
color is due to the cleavage of X-gal by the LacZ gene product,
.beta.-galactosidase. The intensity of the blue color is measured
by colorimetry and quantified with OD units. The OD units are
directly proportional to the activity of the caspase in the system.
The method of quantification is referred to as the "readout".
[0010] In a "Single Component" system an exogenous caspase is
stably over-expressed and autoactivated in a yeast strain
containing the transmembrane receptor, CD95/Fas, ("chimera"). The
chimera is expressed such that most of its cytosolic domain is
replaced by a chimeric transcription factor comprised of the
DNA-binding domain of LexA and the transactivation domain of B42.
Between Fas and the chimeric transcription factor (LexA-B42)
domains are various numbers of tetrapeptide sequences known to be
recognized and cleaved by various members of the Caspase family.
Each genetic system of this disclosure contains an appropriately
matched set of the appropriate caspase to the appropriate chimera.
The activity of the entire system is monitored by the readout.
[0011] An embodiment of this disclosure provides for a method using
a single-component system exemplified above for drug screening
assays wherein the activities of Caspase 1, 2, 3, 4, 5, 6, 7, and 9
are measured and compounds that inhibit Caspase 1, 2, 3, 4, 5, 6,
7, and 9 are identified.
[0012] Furthermore, an embodiment of this disclosure provides for a
method using a single-component system exemplified above for drug
screening assays which may utilize any other substrate of the LacZ
gene, beta-galactosidase, for quantification.
[0013] In a "Two-Component" system an exogenous caspase is
expressed at a low level to maintain its inactive pro-caspase form
along with its exogenous cogent upstream activator. The level of
expression is engineered to prevent autoactivation such that the
interaction of the upstream activator with the pro-caspase results
in the activated caspase and the readout. The level of expression
is engineered to impact assay performance by manipulating several
variables of vector construction, including promoter strength,
promoter inducibility, vector copy number, and number of operators
(transcription factor binding sites) in a reporter gene.
[0014] An embodiment of this disclosure provides for a method using
a two-component system exemplified above for drug screening assays
where the activities of ASC, RAIDD, FADD, Apaf, and active
caspase-9 are measured and compounds that inhibit the interaction
or activity of ASC with pro-caspase-1, RAIDD with pro-caspase-2,
FADD with pro-caspase-8 or pro-caspase-10, an active mutant of Apaf
with pro-caspase-9, active caspase-9 with pro-caspase-3 or
procaspase-7 are identified.
[0015] An embodiment of this disclosure provides for a method using
a two-component system exemplified above for drug screening assays
where the activities are used to identify or discover other or
novel upstream activators of pro-caspase-1, pro-caspase-2,
pro-caspase-3, pro-caspase-4, pro-caspase-5, pro-caspase-6,
pro-caspase-7, pro-caspase-8, pro-caspase-9, and pro-caspase-10 via
the introduction of a cDNA library or other expression conveyance
to screen for biomolecular activators of the pro-caspases.
[0016] Furthermore, an embodiment this disclosure provides for a
method using a two-component system exemplified above for drug
screening assays which may utilize any other substrate of the LacZ
gene, beta-galactosidase, for quantification
[0017] In a "Plural-Component" system an exogenous caspase is
expressed in its inactive pro-caspase form along with its exogenous
cogent activation pathway components consisting of two or more
additional upstream proteins required for activation of the caspase
and the subsequent readout. The levels of expression of the
components are engineered to prevent caspase activation unless all
of the components are present.
[0018] An embodiment of this disclosure provides for a method using
a plural-component system exemplified above for drug screening
assays where the activities of FAS, FADD, DR5, ASC, or NALP1 are
measured and compounds that inhibit the interaction or activity of
FAS with FADD, DR5 with FADD, FADD with pro-caspase-8 or
pro-caspase-10, ASC with NALP1, or NALP1 with pro-caspase-1.
[0019] An embodiment of this disclosure provides for a method using
a plural-component system exemplified above for drug screening
assays where the activities are used to identify or discover other
or novel upstream activators of FADD and NALP1 via the introduction
of a cDNA library or other expression conveyance to screen for
biomolecular activators of FADD or NALP1.
[0020] Furthermore, an embodiment this disclosure provides for a
method using a plural-component system exemplified above for drug
screening assays which may utilize any other substrate of the LacZ
gene, beta-galactosidase, for quantification.
[0021] Another embodiment of the present disclosure incorporates
the use of two-component and/or plural-component systems and by
extension their specific examples to identify caspase activation
protease networks in yeast, and to isolate and study an exogenous
protease network in a cellular context.
[0022] An embodiment disclosed includes methods of performing
chemical library screens to validate the performance of the assays
and to further identify caspase activation protease networks in
yeast, and to isolate and study an exogenous protease network in a
cellular context. The screens are conducted such that all assay
components may be added in automated fashion using integrated
robotic liquid handling systems, moving the plates initially into
carousels that hold .about.180 plates at room temperature, and then
manually applying seals (breathable sealing film from Axygen
Scientific) to reduce evaporation, and moving the bar-coded plates
to a 30.degree. C. incubator for the required time, generally 2-4
days. Each assay plate will contain a row of positive (min) and a
row of negative (max) controls that do not receive compounds but
that receive DMSO in volumes equivalent to the amount of DMSO in
which compounds will be supplied. At the conclusion of the 72 hrs
incubation, plates are robotically delivered to one of the
integrated multi-purpose plate readers for reading at OD620 nm.
Programmable robotic workstations sequence the additions of
reagents, minimizing variations in incubation times. Data from
primary screens are uploaded directly from plate readers into
computers with customized Microsoftexcel software, set up to
calculate Z' factor for each plate, and with hit determinations set
at 50% of the mean value for the negative control values.
[0023] An embodiment disclosed utilizes library screens which may
be performed at several different concentrations (eg., 20, 10, and
5 .mu.M) to compare the hit rates, and to empirically determine an
acceptable concentration for conducting large-scale library
screens. It is often best to employ as high a concentration of
compounds as possible to maximize the chance of identifying active
compounds (avoiding false-negatives), but balance that against an
excessive frequency of hits (avoiding false-positives). A general
rule of thumb is to empirically adjust compound screening
concentrations to achieve a hit rate of 0.1-0.5%. Thus, before
undertaking starting a large library screen, pilot experiments may
be utilized to optimize results such as empirically determining the
effects of DMSO on assay performance, through pilot experiments
where increasing concentrations of DMSO (from 1-10% volume) are
added to the positive and negative controls and the impact on assay
signal and stability is determined. Second, when progressing with
larger libraries (e.g., 50K Chembridge), it is useful to determine
the stability of the positive and negative controls from plate to
plate, assessing the assay performance as time is extended from
minutes to hours, and calculating Z' for each plate as the quality
of the assay's performance is assessed in true screening mode.
[0024] For HTS assays we have measured .beta.-galactosidase
produced by yeast carrying the caspase-cleavable reporter proteins,
and assayed the calorimetric product (OD620 nm) derived from X-gal
substrate in 384 well plates, as a measure of the lac Z reporter
gene activity.
[0025] An embodiment of the present disclosure comprises a high
throughput screening of lac-Z reporter gene activity by measuring
.beta.-galactosidase produced by yeast carrying caspase-cleavable
reporter proteins; and assaying the calorimetric product derived
from x-gal substrate in 384 well plates, at an optical density of
620 nm.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 Illustrates the Typical Prodomain Size of Upstream
and Downstream Caspases.
[0027] FIG. 2 Consisting of FIG. 2A and FIG. 2B is a schematic
representation of an embodiment of the present disclosure using a
single-component system as a genetic system for monitoring caspase
1 activity in yeast cells.
[0028] FIG. 3 Demonstrates the resulting substrate specifities of
the caspases expressed in yeast when using a single-component
system.
[0029] FIG. 4 Consisting of FIG. 4A and FIG. 4B is a schematic
representation of an embodiment of the present disclosure using a
two-component system for caspasel activators in yeast cells.
[0030] FIG. 5 Consisting of FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D,
FIG. 5E, FIG. 5F and FIG. 5G demonstrates the resulting caspase
activation in yeast when using a two-component system.
[0031] FIG. 6 Consisting of FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D)
is a schematic representation of an embodiment of the present
disclosure using a plural-component system for caspase-8 activation
in yeast cells.
[0032] FIG. 7 Consisting of FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D,
FIG. 7E, FIG. 7F and FIG. 7G demonstrates the resulting caspase-8
and caspase-10 activation in yeast when using a plural-component
system.
[0033] FIG. 8 Consisting of FIG. 8A, FIG. 8B and FIG. 8C
demonstrates the basis for the cleavable reporter assay when using
of a two-component and/or plural component system when screening
for NALP1.
[0034] FIG. 9 Consisting of FIG. 9A, FIG. 9B and FIG. 9C
demonstrates the basis for the cleavable reporter assay when using
of a two-component and/or plural component system when screening
for NLRs.
[0035] FIG. 10 Demonstrates the optimization of signal:noise ratio
in microtiter plates when performing high throughput screening of
cDNA libraries with the two or plural-component systems.
[0036] FIG. 11 Consisting of FIG. 11A, FIG. 11B and FIG. 11C
demonstrates the validation of single, two, and plural-component
yeast-based Caspase assays using pharmacological inhibitor of
Caspases.
[0037] FIG. 12 Consisting of FIG. 12A, FIG. 12B and FIG. 12C
demonstrates HTS Implementation of yeast-based NALP1 inflammasome
assay. (A) NALP1 yeast screen controls, and a Yeast Screen of a
LOPAC compound library for NALP1.
[0038] FIG. 13 Consisting of FIG. 13A, FIG. 13B and FIG. 13C
demonstrates the resulting substrate sequence- and Caspase
activity-dependent cleavage of S1, S2, and S9.
[0039] FIG. 14 Demonstrates a Flow chart for cDNA library screening
using a reporter gene strategy based on cleavable transcription
factor using a S1 substrate.
[0040] FIG. 15 Demonstrates a Flow chart for cDNA library screening
using a reporter gene strategy based on cleavable transcription
factor using a S3/S7 substrate.
[0041] FIG. 16 Consisting of FIG. 16A and FIG. 16B demonstrates the
resulting substrate specifities of the caspases expressed in yeast
when using a single-component system.
[0042] FIG. 17 Consisting of FIG. 17A and FIG. 17B demonstrates the
resulting Validation of yeast-based assays for effector Caspase
activators--two and three-component systems.
[0043] FIG. 18 Consisting of FIG. 18A, FIG. 18B, FIG. 18C and FIG.
18D demonstrates the resulting validation of a yeast-based assays
for effector Caspase activators when using a two component
systems.
[0044] FIG. 19 Demonstrates the resulting specificity of upstream
activators of initiator Caspases when using a 2-component
system.
[0045] FIG. 20 Consisting of FIG. 20A and FIG. 20B demonstrates the
resulting validation of a 3-component yeast-based Caspase assay
reconstituting DISC.
[0046] FIG. 21 Demonstrates a Flow chart for cDNA library screening
using 3 component system -application to death receptor
cloning--Example Screening Strategy #1.
[0047] FIG. 22 Demonstrates a Flow chart for cDNA library screening
using 3 component system--application to death receptor
cloning--Example Screening Strategy #2.
[0048] FIG. 23 Demonstrates a Flow chart for cDNA library screening
using 3 component system--application to death receptor
cloning--Example Screening Strategy #3.
[0049] FIG. 24 Consisting of FIG. 24A and FIG. 24B demonstrates a
schematic representation of a 3-component system used for cloning
adapter protein that links Fas to pro-Caspase-10.
[0050] FIG. 25 Consisting of FIG. 25A and FIG. 25B demonstrates the
resulting validation of adapter protein cloning system for
Fas/FADD/Caspase-8/10: Reconstituted DISC.
[0051] FIG. 26 Demonstrates a Flow chart for cDNA library screening
using 3-component system to clone adapters.
[0052] FIG. 27 Consisting of FIG. 27A, FIG. 27B and FIG. 27C
demonstrates the resulting validation the cDNA cloning results in
which the clones that activated the reporter genes were
characterized by recovery of cDNA library plasmids and
retransformation into yeast expressing Fas and pro-Caspase-10.
[0053] FIG. 28 Consisting of FIG. 28A, FIG. 28B and FIG. 28C
graphically depicts the optimization of signal:noise ratio in
microtiter plates using cell density.
[0054] FIG. 29 Consisting of FIG. 29A and FIG. 29B graphically
depicts the optimization of signal:noise ratio in microtiter plates
using time and X-gal concentration.
[0055] FIG. 30 Consisting of FIG. 30A, FIG. 30B, FIG. 30C and FIG.
30D demonstrates a diagram of plasmids for co-expression of
Caspases and substrate cleavable transcription factors in
yeast.
[0056] FIG. 31 Consisting of FIG. 31A, FIG. 31B, FIG. 31C and FIG.
31D demonstrates a diagram of plasmids for expression of upstream
activators of Caspases and lacZ reporter gene in yeast.
[0057] FIG. 32 Consisting of FIG. 32A, FIG. 32B and FIG. 32C
demonstrates a diagram of plasmids for functional screening of cDNA
libraries and expression of upstream activators of Caspases.
DETAILED DESCRIPTION OF THE INVENTION
[0058] Proteolytic processing of proteins is an irreversible
post-translational modification of importance for a wide-variety of
biological processes, including cell division, cell death, cell
differentiation, innate immunity, host-pathogen interactions, and
intracellular protein sorting and trafficking (Lopez-Otin C, and
Overall CM. Protease degradomics: a new challenge for proteomics.
Nat Rev Mol Cell Biol 2002; 3(7): 509-19). Consequently, proteases
have emerged as promising targets for drug discovery for a wide
variety of human diseases, including cancer, neurodegeneration,
ischemic diseases, inflammation, and infectious diseases.
[0059] Proteases can be found in either intracellular or
extracellular (or cell surface) locations, where they encounter
their specific substrates. Among the intracellular families of
proteases are Caspases, Calpains, Deubiquitinating enzymes and
their homologs, and Separase. Still other proteases may have dual
intracellular and extracellular lives. For example, Cathepsins are
normally stored in lysosomes undergoing vesicular recycling between
extracellular and intracellular compartments, but also are released
into the cytosol under various pathological circumstances,
promoting cell death (Cirman T, et al. Selective disruption of
lysosomes in HeLa cells triggers apoptosis mediated by cleavage of
Bid by multiple papain-like lysosomal cathepsins. J Biol Chem 2004;
279: 3578-87).
[0060] From the standpoint of high-throughput screening (HTS) for
identifying chemical inhibitors of proteases, many endopeptidases
(or at least their catalytic domains) can be readily produced in
large quantities by recombinant DNA technology, purified, and
formatted for HTS using convenient fluorigenic or colorimetric
substrates. However, such assay formats have several limitations.
First, standard HTS configurations for proteases are typically
amenable only to screens for inhibitors, not activators of
proteases. Second, assays requiring large amounts of purified,
active protease can be hampered by difficulties in producing by
recombinant methods or purifying from endogenous sources sufficient
amounts of material, as well as by instability problems where
purified proteases lose activity in vitro. Third, because one is
often limited to using only the catalytic domain due to
difficulties of expressing and purifying intact full-length
proteases, opportunities to discover allosteric modulators of
proteases are limited, with most of the standard assays limited to
identification of compounds that target the active site in a
competitive fashion. Fourth, when using single target systems
consisting of purified protease or catalytic domain of protease,
opportunities for identification of compounds that target other
proteins involved in protease activation are lost. Fifth, several
classes of intracellular endopeptidases exists as large families of
closely related enzymes with structurally very similar active
sites, making it difficult to achieve selective inhibitors in the
absence of more context-dependent, multi-component systems that
would provide the opportunity for selective inhibition. Sixth, some
proteases are not active in purified form, requiring necessary
cofactors (e.g. Separins involved in chromosome segregation and
cell division) or requiring membranes (e.g. Presenilins (such as
.gamma.-secretase) involved in Amyloid-beta-peptide
processing).
Caspases
[0061] Caspases are intracellular cysteine proteinases with
specificity for aspartic acid residues in the P.sub.1 position of
substrates. These proteases are well conserved throughout animal
evolution, where they play essential roles in programmed cell death
(apoptosis). In Caenorhabiditis elegans, for example, all
programmed cell deaths that occur during the development of this
simple animal depend upon the presence of an intact CED-3 gene,
which encodes a Caspase (Yuan J, et al., The C. elegans cell death
gene ced-3 encodes a protein similar to mammalian interleukin-1
beta-converting enzyme. CELL 1993; 75: 641-52). In humans, 12 genes
encoding Caspases or Caspase-like proteins have been identified, 10
of which have been clearly and convincingly shown to possess
protease activity and all showing absolute dependence on aspartic
acid at the P1 position of substrates, including Caspases-1-10.
Another member of the family, Caspase-12, is produced only in
.about.1% of African populations, due to a polymorphism that
produce a nonsense mutation that truncates the protein in most
humans, and its proteolytic activity is questionable (Fischer H, et
al., Human caspase-12 has acquired deleterious mutations. Biochem
Biophys Res Comm 2002; 293: 722-6; and Saleh M, et al., Enhanced
bacterial clearance and sepsis resistance in caspase-12-deficient
mice. Nature 2006; 440: 1064-8). Protease activity for the
remaining member, Caspase-14, has not been demonstrated to date
(Kuechle M K, et al., Caspase-14, a keratinocyte specific caspase:
mRNA splice variants and expression pattern in embryonic and adult
mouse. Cell Death Differ 2001; 8: 868-70). In addition to
apoptosis, other biological roles for Caspases have been
elucidated, the most studied of which is inflammation. Of the 10
established human Caspases, 3 of them cleave and activate
pro-inflammatory cytokines (particularly pro-IL-1b, pro-IL-18,
pro-IL-32), having major roles in host-defense and inflammation
(Reed J C. Caspases and cytokines: roles in inflammation and
autoimmunity. Adv Immunol 1999; 73 :265-99).
[0062] Caspases exist as inactive zymogens in all animal cells but
can be activated by proteolytic cleavage of their proforms at
conserved aspartic acid residues, thus generating the subunits of
the enzymatically active proteases which consist of heterotetramers
comprised of two large and two small subunits. Because caspases
both cleave substrates at Asp residues and are themselves activated
by cleavage at Asp residues, the potential for proteolytic cascades
exists and indeed has been documented (reviewed in (Salvesen G S,
et al., Intracellular signaling by proteolysis. Cell 1997; 91
:443-6; and Thornberry N A, and Lazebnik Y. Caspases: enemies
within. Science 1998; 281: 1312-6). The concept of upstream
initiator and downstream effector caspases that operate within a
proteolytic cascade thus has emerged (Salvesen G S, et al., Cell
1997; 91: 443-6; and Alnemri E S, et al. Human ICE/CED-3 Protease
Nomenclature. Cell 1996; 87: 171).
[0063] FIG. 1 helps illustrate that relevant to their mechanisms of
activation, all of the upstream initiator Caspases contain large
prodomains and many of these prodomains have been shown to bind
other proteins involved in triggering the cascade. The downstream
Caspases, which function as the ultimate effectors of apoptosis,
uniformly possess small prodomains and are probably activated
predominantly if not exclusively by proteolytic cleavage by
upstream Caspases. The irreversible cleavage of specific protein
substrates in cells by these downstream effector Caspases is what
directly or indirectly then accounts for the biochemical and
morphological changes that are recognized as apoptosis. Among the
10 established human Caspases, only 3 have small prodomains and are
considered downstream executioners (Caspases-3, 6, 7). The other 7
members of the family have large N-terminal prodomains containing
signature domains involved in protein-protein interactions that
provide the basis for initiating the proteolytic pathway.
Substrate Specificity of Caspases
[0064] The 3D-structure of the active sites of most human Caspases
accommodates tetrapeptide substrates, in which Aspartic acid at the
P1 position is invariant. In Drosophila, Glutamic acid can also be
tolerated for some of the Caspases (Hawkins C J, et al., The
drosophila caspase DRONC cleaves following glutamate or aspartate
and is regulated by DIAP1, HID; and GRIM. J Biol Chem 2000; 275:
27084-93). Differences in the geometry and side-chains of amino
acids surrounding the active site dictate preferences among
Caspases for different tetrapeptide substrates. Surveys of peptide
libraries have elaborated activity profiles for most of the human
Caspases (Talanian R, et al. Substrate specificities of caspase
family proteases. J Biol Chem 1997; 272: 9677-82), as summarized in
Table 1. These peptidyl substrate preferences match the known
cleavage sites of endogenous proteins that are hydrolyzed by
specific Caspases. For example, the WEHD sequence preferred by the
inflammatory Caspase is found in pro-IL-1b and pro-IL-18, while the
DEVD sequence preferred by downstream apoptotic proteases
Caspases-3 and -7 is found in enzymes responsible for the
characteristic chromatin condensation and DNA fragmentation
associated commonly with apoptosis (Timmer J C, and Salvesen G S.
Caspase substrates. Cell Death Differ 2007; 14(1): 66-72).
TABLE-US-00001 TABLE 1 Caspase Substrate Cleavage site Effect
Caspase 3 and 7 Consensus DEVD/G ICAD DETD/S Initiates DNA
fragmentation PARP DEVD/G Inactivates DNA repair PKC.delta. DMQD/N
Activates kinase MEKK-1 DTVD/G Activates kinase U1-70kD DPGD/G
Inactivates mRNA splicing Caspase 6 Consensus VEID/G Lamin A VEID/N
Nuclear collapse Keratin 18 VEVD/A Cytoskeletal collapse Caspase 8
Consensus IETD/G procaspase 3 IETD/S Activates caspase procaspase 7
IQAD/S Activates caspase BID LQTD/G Cytochrome c release Caspase 1
Consensus WEHD/G proIL-1.beta. YVHD/A Activates IL-1.beta. proIGIF
(IL-18) LESD/N Activates IGIF
Mechanisms of Activating Upstream Initiator Caspases
[0065] One of the major pathways for Caspase activation involves
the TNF family of cytokine receptors (reviewed in Wallach D, et
al., Cell death induction by receptors of the TNF family: towards a
molecular understanding. FEBS Lett 1997; 410: 96-106, and Ashkenazi
A, and Dixit V M. Death receptors: signaling and modulation.
Science 1998; 281: 1305-8). Several TNF family receptors are known
to transduce signals that result in apoptosis, including
TNF-R1(CD120a), Fas (CD95), DR3 (Wsl-1; Tramp); DR4 (Trail-R1); DR5
(Trail-R2); and CAR-1. These death receptors contain a conserved
cytosolic domain known as a Death Domain (DD) that is responsible
for recruiting adapter proteins such as Fadd/Mort-1 to the receptor
complex after binding of ligand. The Fadd/Mort-1 protein contains
both a DD domain and an additional protein-interaction domain
called a Death Effector Domain (DED). The DED of Fadd/Mort-1 binds
certain caspases which contain homologous DEDs within their
prodomains, caspases-8 and -10. The oligomerization of caspases
within the death receptor complex results in trans-processing of
the zymogens (Muzio M, et al., An induced proximity model for
caspase-8 activation. J Biol Chem 1998; 273: 2926-30; and Yang X,
et al., Autoproteolytic activation of pro-caspases by
oligomerization. Mol Cell 1998; 1: 319-25), which contain low
levels of proteolytic activity even before undergoing processing to
the fully active protease. Processing of caspase-8 removes the
DED-containing prodomain, thus releasing the activated protease
into the cytosol where it can cleave and activate other downstream
pro-caspases such as caspase-3 (Stennicke H R, et al. Pro-caspase-3
is a major physiologic target of caspase-8. J Biol Chem 1998; 273:
27084-90).
[0066] The pathway for apoptosis induction employed by the TNF
family death receptors is sometimes referred to as the "Extrinsic
Pathway." This pathway for caspase activation plays a critical role
in the mechanisms used by immune cells to induce apoptosis in
target cells. Cytolytic T-cells, for example, employ death ligands,
particularly Fas-Ligand (Fas-L), as a weapon for inducing apoptosis
in target cells (Nagata S, and Golstein P. The Fas death factor.
Science 1995; 267(5203): 1449-56). Several anti-apoptotic proteins
that contain Death Effector Domains have also been described that
operate as suppressers of apoptosis induced by TNF-family death
receptors. These proteins contain DEDs, which allow them to
interact with other DED-proteins, such as Fadd, pro-caspase-8, and
pro-caspase-10, thereby interfering with assembly of the
multiprotein complexes required for death receptor-mediated
activation of caspases (Irmler M, et al. Inhibition of death
receptor signals by cellular FLIP. Nature 1997; 388: 190-5; and
Tschopp J, et al., Inhibition of Fas death signals by FLIPs. Curr
Opin Immunol 1998; 10: 552-8). For example, the cellular protein
Flip (reviewed in Wallach D. Placing death under control. Nature
1997; 388: 123-6) is such an anti-apoptotic protein, representing a
homologue of caspases-8 and -10 that contains DED domains but lacks
proteolytic activity.
[0067] Standing apart from the Death Receptors is another mechanism
for achieving initiator Caspase activation that relies on
nucleotide-binding oligomerization domains in Caspase-binding
proteins to assemble multi-protein complexes, sometimes referred to
as "apoptosome" or "inflammasomes", depending on whether the
Caspases activated are involved in cell death versus inflammation.
The prototype for this Caspase activation mechanism comes from
pioneering work in Caenorhabiditis elegans, where the only
documented mechanism of inducing caspase activation depends on
CED-4. The CED-4 protein is an ATP-binding protein and putative
ATPase, which binds to pro-CED-3, an initiator caspase (Seshagiri
S, and Miller L K. Caenorhabditis elegans CED4 stimulates CED-3
processing and CED-3-induced apoptosis. Curr Biol 1997; 7: 455-60;
and Chinnaiyan A, et al., Role of CED-4 in the activation of CED-3.
Nature 1997; 388: 728-9). Binding of ATP to CED4 induces
conformational changes that result in oligomerization of CED-4
proteins, followed by binding to pro-CED-3 (Yang X, et al.,
Essential role of CED-4 oligomerization in CED-3 activation and
apoptosis. Science 1998; 281: 1355-7). The oligomerization of
associated pro-CED-3 proteins is thought to bring these zymogens
into close proximity, allowing them to dimerize and become active,
typically cleaving themselves or trans-processing each other,
thereby generating the fully active autonomous proteases. The first
mammalian homologue of CED-4 identified was Apaf-1 (Apoptotic
Protease Activating Factor-1). The human Apaf-1 protein shares in
common with CED-4 the presence of a nucleotide binding NB-ARC
domain and a caspase-binding CARD (CAspase Recruitment Domain)
domain. However, Apaf-1 is structurally more complex than the worm
CED-4 (Zou H, et al., Apaf-1, a human protein homologous to C.
elegans CED-4, participates in cytochrome c-dependent activation of
caspase-3. Cell 1997; 90: 405-13), in that it also contains 12-14
copies of a W) domain at its C-terminus. The WD domains function as
negative-regulatory region, rendering Apaf-1 inactive until bound
by cytochrome c from mitochondria (Li P, el al. Cytochrome c and
dATP-dependent formation of Apaf-1/Caspase-9 complex initiates an
apoptotic protease cascade. Cell 1997; 91: 479-89). The presence of
this negative-regulatory domain thus defines a fundamental
difference between the human Apaf-1 and worm CED-4 proteins. The
human protein requires an activation step to interact with caspases
and induce cell death, whereas the worm CED-4 protein has
constitutive caspase-binding and death-inducing activity (reviewed
in Reed J C. Cytochrome C: Can't live with it; Can't live without
it. Cell 1997; 91 :559-62). The only known mechanism for activating
Apaf-1 is cytochrome c, which binds to Apaf-1 apparently relieving
the repression applied by the WD domains (Li P, et al. Cell 1997;
91: 479-89). Cytochrome c is normally sequestered inside
mitochondria, between the inner and outer membranes of these
organelles. However, it becomes released into the cytosol following
exposure of cells to a variety of pro-apoptotic stimuli, including
chemotherapeutic drugs (Liu X, et al., Induction of apoptotic
program in cell-free extracts: requirement for dATP and Cytochrome
C. Cell 1996; 86: 147-57; Kluck R M, et al., The release of
cytochrome c from mitochondria: a primary site for Bcl-2 regulation
of apoptosis. Science 1997; 275 :1132-6; and Yang J, et al.,
Prevention of apoptosis by Bcl-2: release of cytochrome c from
mitochondria blocked. Science 1997; 275 :1129-32). The apical
caspase in the cytochrome c/mitochondrial pathway is pro-caspase-9.
Pro-caspase-9 possesses an N-terminal CARD domain that allows it to
bind the CARD domain of Apaf-1. Oligomerization of Apaf-1 induced
by cytochrome c and ATP (or dATP) brings associated pro-caspase-9
zymogens into close proximity, allowing them to dimerize and become
active (Zou H, et al., An APAF-1 cytochrome c multimeric complex is
a functional apoptosome that activates procaspase-9. J Biol Chem
1999; 274: 11549-56; and Saleh A, et al., Cytochrome c and
dATP-mediated oligomerization of Apaf-1 is a prerequisite for
procaspase-9 activation. J Biol Chem 1999; 274: 17941-5). The
caspase-activation pathway mediated by Apaf-1 as a result of
cytochrome c release from mitochondria is sometimes referred to as
the "Intrinsic Pathway." The complex of Apaf1/cytochrome
c/Caspase-9 is known as the "apoptosome" a donut-like structure
with two stacked rings, each ring having stoichiometry of 7:14:7,
based on high resolution cryo-electron microscopy imaging studies
and other methods (Salvesen G S, and Renatus M. Apoptosome: The
seven-spoked death machine. Develop Cell 2002; 2: 256-7).
[0068] An analogous system for achieving initiator Caspase
activation has been described for inflammatory caspases, in which
members of the NLR family ("NACHT+LRR" proteins, also known as
"NOD-Like Receptors") oligomerize to form a platform for
recruitment and activation of inflammatory caspases such as
Caspase-1, 4, and 5 in humans (reviewed in Martinon F, et al., The
Inflammasome: A molecular platform triggering activation of
inflammatory caspases and processing of proIL-b. Mol Cell 2002; 10:
417-26). NLRs consist of a central nucleotide-binding domain called
NACHT, flanked on the C-terminus by Leucine Rich Repeats (LRRs) and
on the N-terminus by either CARDs or PYRIN Domains (PYDs). The LRRs
of NLRs keep these proteins in an inactive monomeric state, until
bound by pathogen-derived products such as components of bacterial
peptidoglycan. In vitro reconstitution studies by our laboratory
have shown that upon binding pathogen-derive ligands, a
conformation change occurs in NLRs that renders the NACHT domain
competent to bind nucleotide triphosphates (NTPs) and undergo
NACHT-dependent oligomerization (Faustin B, et al. Reconstituted
NALP1 inflammasome reveals two-step mechanism of Caspase-1
activation. Molecular Cell 2007; 25(5): 713-24). The N-terminal
CARD and PYD domains of NLRs bind directly to the CARD of
inflammatory caspases or indirectly link to them via the bipartite
adapter protein ASC, which contains both a CARD and PYD (Stehlik C,
and Reed J C. The PYRIN connection: novel players in innate
immunity and inflammation. J Exp Med 2004; 200: 551-8). The complex
of NLR/ASC/Caspase-1 is called the "inflammasome" (Martinon F, and
Tschopp J. NLRs join TLRs as innate sensors of pathogens. Trends
Immunol 2005; 26: 447-54). Cryo-EM imaging studies suggest that the
NALP1 (NLRP1) inflammasome is a donut-like structure, akin to the
apoptosome (Faustin B, et al. Molecular Cell 2007; 25(5):
713-24).
[0069] Other mechanisms of mammalian Caspase activation have also
been described, including the PIDDosome, a multiprotein complex
involving the p53-inducible protein PIDD, the adapter protein
RAIDD, and Caspase-2 (Tinel A, and Tschopp J. The PIDDosome, a
protein complex implicated in activation of caspase-2 in response
to genotoxic stress. Science 2004; 304: 843-6). PIDD contains an
oligomerization domain and Death Domain (DD). The DD of PIDD binds
a complementary DD found in the bipartite adapter protein RAIDD,
which contains a DD and CARD. The CARD of RAIDD binds the CARD of
pro-Caspase-2, thus providing the necessary link to the
protease.
Phenotypes of Animal Caspases Expressed in Yeast
[0070] The phenotypes of animal Caspases expressed in yeast have
been explored. (Kamada S, et al. A cloning method for caspase
substrates that uses the yeast two-hybrid system: Cloning of the
antiapoptotic gene gelsolin. Proc Natl Acad Sci USA 1998; 95:
8532-7; Hawkins C J, et al., A cloning method to identify caspases
and their regulators in yeast: Identification of Drosophila DIAP1
as an inhibitor of the Drosophila caspase DCP-1. Proc Natl Acad Sci
USA 1999; 96: 2885-90; Kang J, et al. Cascades of mammalian caspase
activation in the yeast Saccharomyces cerevisiae. J Biol Chem 1999;
274: 3189-98; Zhang H, and Reed J C. Studies of apoptosis proteins
in yeast. In: Schwartz L, Ashwell, editors. Methods in Cell
Biology. 2nd ed: Academic Press; 2001. p. 453-68; and Jin C, and
Reed J C. Yeast and apoptosis. Nature Rev Mol Cell Biol 2002; 3:
453-9). Several lessons have been learned from these studies.
First, over-expression of some zymogen forms of Caspases in yeast
is sufficient to result in their activation, particularly the
initiator Caspases, where it is likely that their CARD or
DED-containing prodomains mediate dimerization when proteins are
over-expressed. Second, some animal Caspases kill yeast, whereas
others do not. Third, for those non-lethal Caspases, it is possible
to devise cleavable reporter systems, particularly transcription
factors that activate reporter genes only when cleaved by Caspases
(Hawkins C J, et al., Proc Natl Acad Sci USA 1999; 96:
2885-90).
[0071] The following examples are intended to illustrate but not
limit the invention.
EXAMPLE 1
Title
Development and Testing of a Cleavable Reporter Gene System for
Assaying Protease Activity in Living Yeast
[0072] A reporter gene system for monitoring protease activity in
living yeast was devised. For this purpose, a Type I transmembrane
receptor (CD95; Fas) 4, most of its cytosolic domain was replaced
by a chimeric transcription factor comprised of the DNA-binding
domain of LexA and the transactivation domain of B42. Between Fas
and the chimeric transcription factor (LexA-B42) was cloned various
numbers of tetrapeptide sequences known to be recognized and
cleaved by various members of the Caspase family. As a control,
constructs were prepared in which the sessile aspartic acid within
the tetrapeptide sequence was replaced with glycine (FIG. 2). These
reporter constructs were expressed from plasmids in autotrophic
yeast mutant strains EGY48 (MAT.alpha. trp1, trp1, his3, ura3,
LexA6op-LEU2) or EGY191 (MAT.alpha. trp1, his3, ura3,
LexA2op-LEU2), containing stably integrated LEU2 (allows grown on
leucine-deficient media when the operator is activated by the
transcriptional activator, LexA-B42) and lacZ (produces
.beta.-galactosidase) reporter genes under control of promoters
containing 1, 2, 4, 6 or 8 copies of the LexA-binding operator
(Estojak J, et al., Correlation of two-hybrid affinity data with in
vitro measurements. Mol Cell Biol 1995; 15: 5820-9).
A Genetic System for Monitoring Caspase 1 Activity in Yeast
Cells.
[0073] The demonstration of a genetic system for monitoring
Caspase-1 activity in yeast cells is shown in FIG. 2 consisting of
2A and 2B. (A) Engineered yeast were created that express a type-1
transmembrane protein (Fas-d-S1-TA) in which the Fas devoid of the
death domain (Fas-d) is followed by a caspasel target site
(S1-WEHD), and a transcriptional activator (TA-consisting of LexA
DNA binding domain and B42 activation domain). LexA operators are
located upstream of LacZ (2 operators) and LEU2 (6 operators)
reporter genes, respectively. The cells expressing
6op-LEU2/2op-LacZ/TEF-Fas-d-S1-TA stimulate caspasel activity
reporter, because expression of active caspase 1 (overexpressing a
full-length caspase-1 construct) results in Fas-d-S1-TA cleavage at
the caspase 1 target site (S1), releasing the transcription
activator (TA), which enters the nucleus and activates LacZ and
LEU2 reporter gene transcription. (B) A version of Fas-d-S1-TA in
which the P1 aspartate is changed to glycine (Fas-d-G1-TA) cannot
be cleaved by active caspasel. The cells expressing
6op-LEU2/2op-LacZ/TEF-Fas-d-G1-TA in which the glycine substitution
is found serve as false-positive reporters for molecules that
activate LacZ and LEU2 gene expressions independent of cleavage at
the caspase 1 target site.
[0074] Caspases are initially produced as inactive zymogens, which
typically undergo proteolytic processing to produce active enzymes
composed in most cases of tetrameric assemblies with two large
(.about.20 kDa) and two small (.about.10 kDa) subunits. To produce
the active form of Caspases in yeast, each full-length Caspase
construct was expressed (for Caspases-1, 2, 3, 4, 5, 6, 7, and 9)
at high levels using plasmids with strong promoters, which results
in `spontaneous" activation of these over-expressed proteases,
generating the active, proteolytically processed forms of these
proteases consisting large and small catalytic subunits. As for
Caspases-8 and 10, however, a small amount of each full-length
Caspase construct was expressed with a large amount of adaptor
protein FADD to make the active forms, because large amounts of
active Caspases-8 or -10 inhibited the yeast cell growth
significantly. These active Caspases were then expressed in
.DELTA.Leu yeast with cleavable reporter constructs containing
various tetrapeptide target sequences. Variables such as the
strengths of the yeast promoters driving expression of the
Fas-LexA-B42 cleavable fusion proteins to optimize signal:noise
ratio were empirically adjusted, so that background (spontaneous)
activation of the LEU2 and lacZ reporter genes was minimal (not
shown), while titrating the number of LexA operators in the
reporter genes to ensure a signal well above background,
empirically determining that 2 LexA operators for lacZ and 6 LexA
operators for LEU2 produced satisfactory results (not shown).
[0075] When plated on complete medium, all .DELTA.Leu transformants
grew, as expected (FIG. 3). However, a selective pattern of growth
was observed for yeast transformants when they were plated on
leucine-deficient medium to test activity of the LexAop-LEU2
reporter gene, along with X-gal substrate to colorimetrically
detect the presence of .beta.-galactosidase activity. In yeast
containing the WEHG control sequence, which lacks the sessile
aspartic acid required for Caspase cleavage, none of the yeast
transformants grew or showed .beta.-galactosidase-positivity,
including Caspase-1 through Caspase-10. Yeast in which the
Fas-LexA-B42 fusion protein contains the WEHD tetrapeptide sequence
grew on leucine-deficient media and showed
.beta.-galactosidase-positivity when co-expressed with active
Caspases-1, 4, and 5, but not Caspases-2, 3, 6, 7, 8, 9, or 10,
perfectly matching predictions of prior studies in which the
optimal tetrapeptide substrate sequences were determined for these
human Caspases (Thornberry N A, et al. A combinatorial approach
defines specificities of members of the caspase family and granzyme
B. J Biol Chem 1997; 272: 17907-11).
[0076] Similarly, yeast containing the DEVD linker in the
Fas-LexA-B42 fusion protein grew on leucine-deficient media and
showed .beta.-galactosidase-positivity when co-expressed with
active Caspase-3 or Caspase-7, but not Caspases-1, 4, 5, 6, 8, 9,
or 10, showing striking preferentiality among Caspases for cleavage
of this reporter protein. Caspase-2 also cleaved the
DEVD-containing reporter protein, which differs only slightly from
the reported optimal substrate sequence of DEHD (Thornberry N A, et
al. B. J Biol Chem 1997; 272: 17907-11). DEHD produced results very
similar to DEVD, consistent with prior substrate specificity
experiments suggesting that the P2 position where H was placed is
the most flexible for Caspases-3 and -7 (Thornberry N A, et al. B.
J Biol Chem 1997; 272: 17907-11). The other tetrapeptide sequences
optimized for Caspase-6 (TEVD), Caspases-8/10 (LETD) and Caspase-9
(LEHD) resulted in less specific patterns of reporter protein
activation, but nevertheless showed selectivity. For example, the
"inflammatory" Caspases (Caspases-1, 4, and 5) did not activate the
Caspase-6 substrate (TEVD). Also, the downstream effector Caspases
(Caspases-3, 6, 7) did not activate the reporter proteins
containing tetratpeptide sequences optimized for upstream initiator
Caspases (LETD, Caspases-8/10; LEHD, Caspase-9).
Substrate Specifities of the Caspases Expressed in Yeast.
[0077] As shown in FIG. 3 versions of Fas-d-S1-TA (Fas-d-G1-TA,
Fas-d-S2-TA, Fas-d-S3-TA, Fas-d-S6-TA, Fas-d-S8-TA, and
Fas-d-S9-TA) were created by substituting the caspasel cleavage
site (S1-WEHD) with the pseudo-site (G1-WEHG), the caspase2
cleavage site (S2-DEHD), the caspase3 cleavage site (S3-DEVD), the
caspase6 cleavage site (S6-TEVD), the caspase8 cleavage site
(S8-LETD), and the caspase9 cleavage site (S9-LEHD), respectively,
and were expressed with the LacZ and LEU2 reporter genes. The
resultant yeast strains EGY48 expressing
6op-LEU2/2op-LacZ/TEF-Fas-d-G1-TA,
6op-LEU2/2op-LacZ/TEF-Fas-d-S1-TA,
6op-LEU2/2op-LacZ/TEF-Fas-d-S2-TA,
6op-LEU2/2op-LacZ/TEF-Fas-d-S3-TA,
6op-LEU2/2op-LacZ/TEF-Fas-d-S6-TA,
6op-LEU2/2op-LacZ/TEF-Fas-d-S8-TA, or
6op-LEU2/2op-LacZ/TEF-Fas-d-S9-TA, were transformed with caspase
expression plasmids. Substrate specificities were determined for
the ten caspases (caspases1-10, C1-C10). If cells express the
active caspases with the suitable cleavage sites, the cells can
grow in the selection medium (without leucine) and they hydrolyze
X-gal to become blue. Expression of the Caspases
(p424-ADH-Caspase1-FLAG, p424-ADH-HA-Caspase2, p424-ADH-Caspase3,
p424-TEF-Caspase4, p424-ADH-Caspase5, p424-TEF-HA-Caspase6,
p424-ADH-Caspase7, p424-CYC1-Caspase8-HA/TEF-HA-FADD,
p424-ADH-HA-Caspase9-FLAG, and pCYC1-Caspase10-HA/TEF-HA-FADD) had
no effects on the cell growth when plated on regulate
(leucine-containing) medium..fwdarw.
[0078] Taken together, these data demonstrate the performance of a
one-component cleavable reporter gene system for measuring activity
of the human Caspases in yeast. This one-component system was
further used for screening human cDNA libraries expressed in yeast
plasmids, where the cleavable Fas-LexA-B42 fusion protein was
expressed without a Caspase, screening cDNA libraries for proteases
that activate the reporter gene. Using the WEHD-containing reporter
protein, eleven cDNAs that confirmed positive on repeated testing
were obtained, including five that encoded Caspase-1 and six
encoding Caspase-4. Using the DEVD-containing reporter, twelve
clones that confirmed positive were obtained, including three that
encoded Caspase-3 and nine encoding Caspase-7. These cDNA library
screening results further validated the cleavable reporter
system.
Development of Two-component Systems Demonstrating Function of
Caspase Activators in Yeast
[0079] Two-component systems for assaying Caspase activity in
yeast, in which the inactive proforms of the Caspases were
co-expressed with various activator proteins were also created.
FIG. 4 depicts the concept for Caspase-1, showing its preferred
substrate WEHD.
Schematic Representation of a Two-component System for Caspase1
Activators in Yeast Cells.
[0080] The schematic representation of a two-component system for
caspase I activators in yeast cells is shown in FIG. 4 consisting
of 4A and 4B (A) The zymogen (inactive) pro-caspase-1 protein is
expressed in yeast, with the cleavable reporter containing the S1
site (WEHD), by regulating the expression level
(p413-TEF-Fas-d-S1-TA/DTEF1-Caspase1-FLAG) in the yeast cell
(EGY191 expressing 2op-LEU2/2op-LacZ). Under these conditions the
reporter genes were silent. (B) Co-expression of ASC at high levels
from p424-TEF-HA-Asc activated Caspase1, resulting in S1 site
cleavage, and the transcription factor was released from the
membrane to enter the nucleus and activate reporter genes.
[0081] Among the two-component systems interrogated were
combinations of upstream initiator Caspases co-expressed with known
activators, including: (1) pro-Caspase-1 plus ASC; (2)
pro-Caspase-2 plus RAIDD; (3) pro-Caspase-9 plus a gain-of-function
mutant of Apaf1; (4) pro-Caspase-8 plus FADD; and (5)
pro-Caspase-10 plus FADD. In each of these cases, the upstream
initiator Caspase, contains a N-terminal pro-domain (either CARD or
DED) that binds a compatible CARD or DED in the activator protein.
A two-component systems involving an upstream and downstream
protease was also tested. For example, Caspase-9 was previously
known as a direct upstream activator of downstream proteases,
Caspases-3 and -7 (Slee E, et al. Ordering the cytochrome
c-initiated caspase cascade: Hierarchic activation of
caspases-2,-3,-6,-7,-8 and -10 in a caspase-9-dependent manner. J
Cell Biol 1999; 144: 281-92). Active Caspase-9 was over-expressed
in combination with the inactive proforms of Caspase-3 or -7,
expressed at low levels. In each case, an appropriate cleavable
reporter protein was co-expressed with the two-component systems,
in yeast containing LEU2 and lacZ reporter genes. For all pair-wise
combinations of pro-Caspase and upstream activator, the pro-Caspase
was expressed in a relatively small amount using a low-copy plasmid
containing CEN/ARS (1 to 2 copies per cell), and the upstream
activator in a large amount using 2.mu. origin-containing plasmids
(20 to 50 copies per cell) with relatively strong promoters, TEF,
or ADH. Each pair of pro-Caspase and upstream activators were
empirically adjusted by manipulating the number of LexA operators
driving the LEU2 or lacZ reporter genes to achieve an acceptable
signal:noise ratio. For example, for the combination of
pro-Caspase-1 and ASC, it was determined that 2 LexA operators for
LEU2 were superior to 4 or 6 operators (FIG. 4).
[0082] Successful activation of the cleavable reporter protein was
achieved for each two-component system (FIG. 5). Expression of
either the pro-Caspase alone or the upstream activator alone failed
to result in activation of the LEU2 and lacZ reporter genes, as
determined by ability to grow on leucine-deficient plates and
.beta.-galactosidase-positivity, confirming that both components
are necessary for activating the reporter proteins. Moreover,
pairing pro-Caspases with the wrong activators (e.g.
Apaf1*+pro-Caspase-8; ASC+pro-Caspase-9) also failed to activate
the reporters (not shown). Taken together, these data demonstrate
the utility of yeast-based protease screening using two-component
systems. Two-component systems for caspase activation in yeast.
[0083] As shown in FIG. 5 consisting of FIGS. 5A-5G yeast
transformants were plated on leucine-deficient medium containing
X-gal. For controls, the "empty" version of the plasmid was always
introduced so that cells were subjected to identical selection
conditions. (A) The recipient yeast strain EGY191 contained
2op-LEU2/2op-LacZ/TEF-Fas-d-S1-TA, with or without plasmids
encoding pro-Caspase-1 (DTEF1-Caspase1-FLAG) or ASC
(p424-TEF-HA-Asc), and cleaved the S1 site, because Asc itself did
not cut the S1 site. (B) The recipient yeast strain EGY48 contained
6op-LEU2/2op-LacZ/DTEF2-Fas-d-S2-TA, with or without plasmids
encoding pro-Caspase-2 (DGPD1-HA-Caspase2-FLAG) or RAIDD
(p424-TEF-HA-RAIDD). (C) The recipient yeast strain EGY48 contained
6op-LEU2/2op-LacZ/GPD-Fas-d-S8-TA, with or without plasmid encoding
pro-Caspase-8 (CYC1-Caspase8-HA) or FADD (p424-TEF-HA-FADD). (D)
The recipient yeast EGY48 contained
6op-LEU2/2op-LacZ/TEF-Fas-d-S9-TA, with or without plasmids
encoding pro-Caspase-9 (pTEF-HA-Caspase9) or an active mutant of
Apaf-1 (p424-TEF-HA-Apaf*). (E) The recipient yeast strain EGY48
contained 6op-LEU2/2op-LacZ/GPD-Fas-d-S8-TA, with or without
plasmids encoding pro-Caspase-10 (ADH-Caspase10-FLAG) or FADD
(p424-TEF-HA-FADD). (F, G) The recipient yeast EGY191 contained
2op-LEU2/2op-LacZ/DTEF3-Fas-d-S3-7-TA, with or without plasmids
encoding Caspase-9 (p424-ADH-HA-Caspase9-FLAG) and pro-Caspase-3
(CYC2-Caspase3) or pro-Caspase-7 (p424-ADH-Caspase7).
Development of a Plural-component System--Mammalian Protease
Activating Pathways Reconstituted in Yeast
[0084] An increase in the complexity of reconstituted
Caspase-activating systems in yeast, was created utilizing a plural
component network comprising three or more components. Two
different mammalian Caspase-activating networks were explored. In
the first, the proximal portion of the extrinsic pathway was
recapitulated, expressing (1) death domain (DD)-containing
TNF-family death receptor, Fas [CD95]; (2) bipartite adapter
protein FADD, which contains DD and DED; and (3) the proform of a
DED-containing protease, either Caspase-8 or Caspase-10, along with
(4) a cleavable reporter protein containing the Caspase-8/10
substrate tetrapeptide LETD. Two strategies for reconstituting the
Fas/FADD/pro-Caspase-8 network in yeast were compared. First, we
expressed the bridging adapter protein FADD at low levels using a
constitutive but weak promoter such that the amount of FADD was
inadequate to achieve pro-Caspase-8 activation in the absence of
Fas. For that system, Fas was expressed at high levels so that it
could oligomerize with itself without requiring Fas Ligand confirm
(FIGS. 7A, and 7B). Second, low levels of pro-Caspase-8 and high
levels of FADD (FIGS. 7C, and 7D) were expressed.
[0085] Additionally a 4-component system was reconstituted in yeast
for .gamma.-secretase activity, comprised of Presenlin-1,
Nicastrin, APH-1, and PEN-2, using lacZ (.beta.-galactosidase)
reporter gene activation by a membrane-tethered cleavable
transcription factor (Amyloid .beta.-protein precursor [APP] fused
to GAL1 transcription factor) as an endpoint for assessing activity
of this transmembrane protease complex.
Schematic Representation of Plural-component Systems for Caspase-8
Activation in Yeast.
[0086] The schematic representation of a plural-component system
for caspase-8 activators in yeast cells is shown in FIG. 6
consisting of FIGS. 6A-D (A) The zymogen pro-Caspase8 (from plasmid
CYC1-Caspase8-HA) was expressed with substrate containing the S8-10
cleavage element (from plasmid p413-TEF-Fas-d-S8-TA) in the yeast
EGY48 expressing 6op-LEU2. A small amount of FADD was also
expressed without activating the Caspase-8 from plasmid
p426-2op-LacZ/DADH1-FADD. (B) Addition of FADD-binding protein Fas,
expressed at high levels from plasmid p424-Fas) activated
Caspase-8, releasing the transcription factor. (C, D) In an
alternative approach, FADD expressed under control of an inducible
plasmid, using p424-ADH-HA-FADD, thus completing the connection
between constitutively expressed pro-Caspase-8 and Fas.
[0087] By empirically comparing different strength promoters for
driving expression of the three components and adjusting the number
of LexA operators in the promoters of the LEU2 and lacZ reporter
genes, the determination of conditions that produced the desired
result for the approach was reached whereby low levels of
constitutive FADD were complemented by expression of Fas
confirm(FIGS. 9A and 9B). The alternative approach resulted in
Fas-independent activation of Caspase-8 and -10, though the
reactions were enhanced by Fas or other death receptors (e.g.
DR5).
Testing of Yeast-based, Plural-component Systems for Activating
Caspases-8 or -10.
[0088] The results of testing yeast-based, plural-component systems
for activating caspases-8 or -10 are shown in FIG. 7 consisting of
FIGS. 7A-G. (A, B) Yeast EGY48 cells expressing
Caspase-8/-10-cleavable substrate from
6op-LEU2/2op-LacZ/TEF-Fas-d-S8-TA, a n d either pro-Caspase-8 or
pro-Caspase-10 (from CYC1-Caspase8-HA or CYC1-Caspase10- FLAG), and
FADD from a plasmid with a weak promoter were transformed with an
empty vector (p424-ADH) or a plasmid expressing Fas (p424-ADH-Fas).
The small amount of pro-Caspase-8 was not activated by the small
amount of FADD, unless Fas was present, resulting in cell growth
with blue color when transformed cells were grown on medium lacking
leucine and containing X-gal substrate. (C, D) Yeast EGY48 cells
expressing Caspase-8/-10-cleavable substrate, low levels of either
pro-Caspase-8 or -10, and either Fas or DR5 were transformed with
an empty vector p424-ADH or the same plasmid expressing FADD
(p424-ADH-HA-FADD) from a strong promoter. In the absence of FADD,
the small amount of pro-Caspase-8 was not activated and did not cut
the S8 site of the Fas-d-S8-TA substrate, but when FADD was
supplied, then substrate cleavage was achieved. However, Caspase-8
and -10 activation were Fas/DR5-independent (constituting a
two-component but not plural or three-component system) but the
reactions were enhanced significantly by expressing Fas or DR5.
Examples of cDNA cloning results. Yeast strain EGY48 containing
6op-LEU2 and 2op-lacZ reporter genes and expressing the
LETD-containing substrate (expressed from pTEF-Fas-d-S8-TA) with or
without pro-Caspase-8 (CYC1-Caspase8-HA), and/or FADD
(.DELTA.ADH1-FADD). Cells were subsequently transformed with
various cDNA libraries (See FIGS. 21-23) and clones that activated
the reporter genes were characterized by recovery of cDNA library
plasmids and retransformation into yeast expressing FADD and
pro-Caspase-8 (E), pro-Caspase-8 without FADD (F), or neither (G).
Among the positive clones were #XA514 (n=13) encoding a fragment of
DR4 (G210-E468), #XA512 (n=15) encoding a fragment of DR5
(A43-S411), #XD108 (n=8) encoding a fragment of DR5 (Y99-S411), and
#XD422 (n=3) encoding a fragment of DR5 (A192-S411), and #XD402
(n=7) encoding a fragment of DR5 (V124-S440), all of which
activated the lacZ reporter gene in cells containing FADD and
Caspase-8 (E) but not in cells lacking FADD (F) or both (G). Also
obtained were cDNAs #XD103 encoding a fragment of Caspase-8
(M1-R233), #X8716 encoding a full-length FLIPL, and #XA411 encoding
a full-length Caspase-2. Assays were performed in duplicate,
growing cells for 4 days on plates.
[0089] Another plural-component Caspase-activating network was
similarly created, using variations on the same approach.
Specifically, the pro-inflammatory plural-component system was
reconstituted in yeast and comprised: (1) the CARD-containing
protease, pro-Caspase-1; (2) the bipartite adapter ASC, which
contains CARD and PYD domains; and (3) the NLR-family protein
NALP1, using a gain-of-function mutant of NALP1 that is
constitutively active without requiring a bacterial ligand to
induce its oligomerization (Faustin B, et al. Reconstituted NALP1
inflammasome reveals two-step mechanism of Caspase-1 activation.
Molecular Cell 2007; 25(5): 713-24). For this system, the reporter
protein contained the Caspase-1 cleavage sequence, WEHD (FIG. 8).
Pro-Caspase-1 and adapter protein ASC was expressed in yeast under
constitutive promoters, using a weak promoter for ASC that failed
to activate pro-Caspase-1 in the absence of other cofactors. The
gain of function mutant NALP1(.DELTA.LRR) was expressed under
control of an inducible promoter. Upon induction of
NALP1(.DELTA.LRR) expression, the reporter genes became activated.
Exclusion of any of the three components of this system (NALP1,
ASC, or pro-caspase-1) prevented reporter gene activation (not
shown). It was determined that NALP1.DELTA.LRR can activate
pro-Caspase-1 in yeast in an ASC-dependent manner, thus
demonstrating reconstitution in yeast of another plural-component
system for Caspase activation.
Yeast Assay for NALP1.
[0090] The results of a yeast assay for NALP1 are shown in FIG. 8
consisting of FIGS. 8A-C. (A, B) The basis for the cleavable
reporter assay is depicted. The membrane tethered transcription
factor consists of the DNA-binding domain of the LexA protein and
the transactivation domain of the B42 protein, fused to a peptide
linker with caspase-1 cleavage sequence (WEHD) and the
extracellular and transmembrane domains of Fas (CD95). In the
absence of caspase-1 activators, the reporter remained uncleaved
and membrane anchored (A). When caspase-1 was activated, then the
reporter was cleaved, releasing the chimeric LexA/B42 transcription
factor to leave the membrane and enter the nucleus, where it
induced expression of LEU2 and LacZ genes (B). (C) An example of
the assay results was shown. Yeast were plated on selective media
and .beta.-galactosidase activity was ascertained by a calorimetric
assay. Plasmids employed were empirically optimized. The example
shown employed EGY48 strain yeast, which contain a LacZ reporter
gene containing two copies of the LexA operator and in which ASC
expression was driven from a plasmid containing a constitutive
promoter with weak activity (CYC1). Two different strength
promoters were tried for expressing pro-caspase-1: P.DELTA.TEF2
(top) and P.DELTA.TEF3 (bottom), both of which worked well for this
assay. The yeast cells were transformed with a GAL1 promoter-driven
plasmid containing no cDNA insert (left) or a cDNA encoding
NALP1.DELTA.LRR (middle). Alternatively, a plasmid with TEF
promoter driving expression of NALP1.DELTA.LRR was employed
(right).
Yeast Assay for NLRs.
[0091] The results of a yeast assay for NLRs are shown in FIG. 9
consisting of FIGS. 9A-C. (A) Yeast strain EGY48 containing
6op-LEU2/2oplacZ expressing pro-Caspase-1 (driven by .DELTA.TEF3
promoter, which expresses at low levels) with membrane tethered
transcription factor substrate containing WEHD peptide linker (S1
substrate) were transformed with plasmids encoding adapter protein
Asc (expressed at low levels from a CYC promoter) and upstream
activators either NLRP1.DELTA.LRR or NLRP3.DELTA.LRR (expressed at
high levels from TEF or GAL1 promoters). When Caspase-1 is
activated via the combination of Asc and NLPR1 or NLRP3, the
reporter is cleaved, releasing the chimeric LexA/B42 transcription
factor to leave the membrane and enter the nucleus, where it
induces expression of LEU2 and lacZ genes. (B) EGY48 cells
(6op-LEU2, 2op lacZ) were transformed with plasmids encoding the S1
substrate, pro-Caspase-1 (C1) expressed from .DELTA.TEF promoter,
Asc (low) from CYC promoter, and NLRP3.DELTA.LRR from TEF promoter.
Control transformants received the corresponding empty plasmids
(-). Cells were grown for 2 days on leucine deficient plates
containing X-gal and galactose (to induce NLRP3.DELTA.LLR
expression). (C) The EGY48 recipient (6op-LEU2, 2op-lacZ) yeast
cell strain contained plasmids encoding substrates with either the
WEHD tetrapeptide cleavable linker (S1 substrate expressed from
TEF-Fas-d-S1 (WEHD)-TA transcriptional unit) or WEHG non-cleavable
substrate (G1 substrate expressed from TEF-Fas-d-G1 (WEHG)-TA) in
plasmids encoding either wild-type ("C1/WT") or Cys285 mutated
Caspase-1 ("C285.fwdarw.G285") (expressed from
p.DELTA.TEF1-Caspase-1-FLAG or .DELTA.TEF1-Caspase-1
(C285.fwdarw.G285)-FLAG transcriptional units). Cells were
transformed with the plasmids expressing a small amount of Asc
(CYC-Asc), a large amount of Asc (TEF-Asc), a small amount of
NLRP1.DELTA.LRR (CYCNLRP1), a large amount of NLRP1.DELTA.LRR
(TEF-NLRP1), various combinations, or empty vectors (-). Yeast
transformants were plated on leucine-deficient medium containing
Xgal. The large amount of Asc (expressed from TEF promoter)
activated Caspase-1 by itself. NLRP1.DELTA.LRR was unable to
activate Caspase-1 by itself, albeit expressed at high levels
(confirmed by immunoblotting [not shown]). NLRP1.DELTA.LRR
activated pro-Caspase-1 in the presence of a small amount of Asc,
under conditions where the amount of Asc expressed (from CYC
promoter) was insufficient to independently activate Caspase-1.
HTS Implementation of Yeast-based Protease Network Reconstitution
Assays.
[0092] When converting the yeast-based protease reporter system to
HTS assay, conditions from agar plates to liquid medium in
microtiter wells must be revised. This revision comprises,
utilizing the measured .beta.-galactosidase produced by yeast
carrying the Caspase-cleavable reporter proteins, and assaying the
colorimetric product (OD620.sub.nm) derived from X-gal substrate in
384 well plates, as a measure of the lacZ reporter gene activity.
We compared .beta.-galactosidase activity produced in the presence
or absence of the broad-spectrum caspase inhibitory compound,
zVAD-fmk (benzoyl-Valinyl-Alaninyl-Aspartyl-fluoromethylketone).
Among the variables that were initially interrogated were initial
seeding cell density, time of culture, concentration of X-gal
substrate, and supporting carbohydrate (raffinose vs mannose). Note
that it was determined that it is not advisible to employ glucose
in yeast media because it represses the GAL1 promoter used in some
plasmids. An example of data obtained using the plural-component
system of Fas/FADD/Caspase-8 is presented. All variables tested
altered the signal:noise ratio of the assay. For the
Fas/FADD/Caspase-8 plural-component assay, the best results were
achieved with .about.2.times.10.sup.5 cells per well starting
density cultured for .about.3 days in raffinose-containing media
with 4.times. concentration of X-gal substrate (FIG. 10). For the
two-component system of ASC/Caspase-1 and for the plural-component
system of NALP1.DELTA.LRR/ASC/Caspase-1, the same conditions were
optimal, among those tested (not shown). For the Fas/Fadd/Caspase
8, the caspase and adaptor protein (Fadd) were expressed at
relatively small amounts (a weak promoter and low copy number
plasmid are used) and the upstream activator (Fas) was expressed in
a large amount.
Optimization of Signal:noise Ratio in Microtiter Plates.
[0093] As shown in FIG. 10 EGY48 cells containing the
plural-component Fas/FADD/Caspase-8 system and Caspase-8-cleavable
reporter were grown in 384 well plates, comparing cell densities,
X-gal concentrations, and sugar additives (mannose vs raffinose).
The activity of .beta.-galactosidase was measured after 72 hrs
culture (mean+std dev; n=3), for cells grown in the absence (white
bars) or presence (black bars) of 100 .mu.M zVAD-fmk Caspase
inhibitor.
[0094] The 384 well assay format was further validated, using
zVAD-fmk, a peptidyl inhibitor with broad-spectrum activity against
animal Caspases. For some experiments, a Calpain inhibitor was
employed in side-by-side experiments as a control. The
broad-spectrum Caspase inhibitor zVAD-fmk inhibited
.beta.-galactosidase activity in a dose-dependent fashion, with the
concentration required for achieving 50% inhibition ranging from
.about.1-10 .mu.M in these assays (FIG. 10). In contrast, the
Calpain inhibitor did not suppress activation of the lacZ reporter
gene. These experiments verified that the 384 well version of the
yeast-based Caspase-dependent reporter gene assay faithfully
indicates the activity of an inhibitory compound, thus fulfilling
one of the prerequisites for developing HTS assays.
Validation of Yeast-based Caspase Assay Using Pharmacological
Inhibitor of Caspases.
[0095] The validation results of a yeast-based Caspase assay using
a pharmacological inhibitor of Caspases are shown in FIG. 11
consisting of FIGS. 11A-C (A) Caspase-1, single-component assay.
Yeast cells expressing TEF-Fas-d-S1-TA/GPD-HA-Caspase1-FLAG were
incubated for 3 days in selection media (SD/X-gal) with the
indicated concentrations of z-VAD-fmk ( ) or DMSO only
(.smallcircle.), using 384-well plates. (B) ASC/Caspase-1
two-component system. Yeast cells expressing
TEF-Fas-d-S1-TA/DTEF1-Caspase1-FLAG/TEF-HA-Asc were incubated for 3
days in the selection media (SD/X-gal) in 384 well plates with the
indicated concentrations of calpeptin (.tangle-solidup.) or
z-VAD-fmk ( ) or an equivalent volume of DMSO was added
(.smallcircle.). (C) Fas/FADD/Caspase8 plural-component system.
Yeast cells expressing TEF-S8-TA/CYC1-caspase8-HA with a small
amount of FADD and a large amount of Fas, were incubated in
selection media (SD/X-gal) with the indicated concentrations of
calpeptin (.tangle-solidup.) or z-VAD-fmk ( ), or equivalent volume
of DMSO (.smallcircle.), in 384-well plates for 3-4 days. For
(A-C), relative absorbance at 620 nm was measured (mean .+-.std
dev; n=3-5 independent experiments). (* indicates p<0.001).
[0096] To assess the reproducibility of the 384 well microplate
assay, multiple replicate wells in 384 well plates were prepared
representing the negative (assay max) and positive (assay min)
controls for the assays. For the plural-component system of
NALP1.DELTA.LRR/ASC/Caspase-1, the maximum signal was produced by
cells grown with X-gal substrate, while the minimum was set by
cells grown without X-gal substrate. As described above, for this
assay, NALP1.DELTA.LRR-mediated activation of Caspase-1 resulted in
activation of the cleavable transcription factor, producing
.beta.-galactosidase, which was measured using a colorimetric
substrate. The magnitude of the readout of the Z' factor was
optimized by empirically adjusting yeast cell density, incubation
time, and other variables in ordere to employ a 384 well assay
format. The Z' factor for the assay was determined by reading
multiple replicates of the assay max and min, and determined to be
>0.6, and thus suitable for HTS (FIG. 12). A screen of 1280
compounds was performed (LOPAC library), achieving an acceptable
average hit rate of .about.0.5 per 384 well plate. An example of
primary screening results is shown for a 384 well plate in which 3
inhibitory compounds were identified (FIG. 12B).
HTS Implementation of Yeast-based NALP1 Inflammasome Assay.
[0097] The results of a HTS of a yeast-based NALP1 inflammasome
assay are shown in FIG. 12 consisting of FIGS. 12A-G (A) NALP1
yeast screen controls. Yeast cells containing
ASC/Nalp1.DELTA.LRR/Caspase1 were plated at 1.88.times.10.sup.5
cells/well in a 384 well plate in media containing 1% galactose/2%
raffinose, with (squares) or without (diamonds) 400 .mu.g/ml X-gal
substrate. The plates were incubated for 72 hrs at 30.degree. C.
and the plates were read at OD620. (B) Yeast Screen of LOPAC
compound library plate 4 for NALP1. Yeast cells containing
ASC/NALP1/Caspase1 were plated at 1.88.times.105 cells/well in a
384 well plate in media containing 1% galactose, 2% raffinose, with
or without 400 .mu.g/ml X-gal. The plates were incubated for 72 hrs
at 30.degree. C. and the plates were read at OD620. The compounds
were screened at 100 .mu.M and "hits" were defined by >50%
reduction (as shown). (C) Validation of HTS assay for
Fas-FADD-Caspase-8 system. Yeast cells containing
Fas/FADD/Caspase-8 were cultured in 384 well plates as above with
(blue squares) or without (purple diamonds) Xgal substrate, the
plates were incubated for 72 hrs at 30.degree. C. and analyzed as
above. (D) Example of library screening plate for
Fas-FADD-Caspase-8. Yeast cells containing Fas/FADD/Caspase-8 were
plated at .apprxeq.2.times.105 cells/wells in 384 well plates in
Selection Media, without (purple triangles) or with (all others)
400 .mu.g/ml X-gal (final concentration), and without (triangles,
squares) or with (diamonds) compounds in 10% DMSO (.about.10 .mu.M
final). Plates were incubated for 48 hrs at 30.degree. C. and OD620
values recorded. "Hits" were defined by >50% reduction (shown as
green diamonds). (E, F) Examples of compounds showing evidence of
selective inhibition of Fas-FADD40 Caspase-8. EGY48 yeast cell
lines employed for deconvoluting hits included yeast expressing
lacZ gene (.quadrature.) expresses LexA/B42 transcription factor
from GAL1 promoter; cells grown in galactose media to induce
promoter) to eliminate false-positives due to .beta.-galactosidase
inhibition and Asc/Caspase-1-expressing yeast (.quadrature.) to
eliminate hits that interfere with a different Caspase. Yeast
containing empty vector were also included (.quadrature.). Yeast
cells (at 2.times.105 cells/well) containing Fas/FADD/Caspase-8
(.tangle-solidup.) or other strains were cultured in 384 well
plates in a total volume of 40 .mu.L Selection Media, with 400
.mu.g/ml X-gal final concentration, without ("C") or with various
concentrations of compound CID-5154 or CID-3101. The plates were
incubated for 2-4 days at 30.degree. C. and OD620 values recorded,
(G) Example of compound showing evidence of inhibition of
Caspase-8. An in vitro biochemical assay was used containing
purified recombinant Caspase-8 and fluorigenic substrate
Ac-IETD-AFC. Data represent relative fluorescence units (RFU)
assessed in the absence or presence of various concentrations of
compound (CID-2531).
Determination of IC50 Values for zVAD-fmk Inhibition of Caspases in
Yeast.
[0098] Yeast expressing various Caspases alone (at high levels) or
in combination with upstream activators (at low levels) and
cleavable substrates containing appropriate tetrapeptides
reorganized by these proteases were used in 384 well
.beta.-galactosidase activity assays to assess inhibition by
zVAD-fmk. The compound was titrated into assays at various
concentrations and percentage inhibition was determined. IC50
values were determined using PRIZM software for analysis with the
results displayed in Table 2 below.
TABLE-US-00002 TABLE 2 IC.sub.50 values for z-VAD inhibition of
Caspases Enzyme IC.sub.50 (uM) Caspase1 3.07 Caspase1 activated
with Asc 1.00 Caspase6 6.78 Caspase7 4.11 Caspase8 activated with
FADD + Fas 5.42 Caspase9 activated with Apaf* 10.66 Caspase10
activated with FADD 9.52
Substrate Sequence- and Caspase Activity-dependent Cleavage of the
S1, S2, and S9.
[0099] As shown in FIG. 13 consisting of 13A, 13B and 13C yeast
transformants were plated on leucine-deficient medium containing
X-gal. (A) The recipient yeast cell strains,
EGY48-6op-LEU2/2op-lacZ/TEFFas-d-S1 (WEHD)-TA (S1), or
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G1(WEHG)-TA (G1) were transformed
with the plasmids encoding the active wild type Caspase1 (WT), the
catalytically-defective Caspase1 (C285.fwdarw.G285), and the empty
vector (-). (B) The recipient yeast cell strains,
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S2(DEHD)-TA (S2), or
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G2(DEHG)-TA (G2) were transformed
with the plasmids encoding the active wild type Caspase2 (WT), the
catalytically-defective Caspase2 (C320.fwdarw.A320), and the empty
vector (-). (C) The recipient yeast cell strain,
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S9(LEHD)-TA (S9), or
EGY48-6op-LEU2/2oplacZ1 TEF-Fas-d-G9(LEHG)-TA (G9), were
transformed with the plasmids encoding the active wild type
Caspase9 (WT), the catalytically-defective Caspase9
(C287.fwdarw.A287), and the empty vector (-).
Flow Chart for cDNA Library Screening Using a Reporter Gene
Strategy Based on Cleavable Transcription Factor.
[0100] The example shown in FIG. 14 is S1 substrate. The yeast
strain EGY48 containing 6op-LEU2 and 2op-lacZ reporter genes and
Caspasecleavable substrate (expressed from TEF-Fas-d-S1-TA) was
transformed (left) with a HEK293 cell cDNA library
(oligo-dT-primed, PGAL1 promoter). Independent colonies of
5.1.times.105 clones appeared on growth plates in 48 hours. Cells
were collected, pooled, and a portion (3.6.times.106) was seeded on
leucine-deficient selection plates containing Xgal. Blue-colored
colonies (n=42) appeared within a week, of which 6 corresponded to
clone #ZB311 encoding full-length Caspase-4 (which is known to
cleave WEHD tetrapeptide [Thornberry, N. A, et. al., J. Biol. Chem.
272, 17907-17911 (1997)]), while the rest were false positives.
(Right) Another cDNA library screen (HEK293 random primed, ADH
promoter used to drive expression) resulted in 2.times.106 clones,
which were pooled and 2.1.times.107 cells were screened on
leucine-deficient, X-gal-containing plates, resulting in 10 blue
colonies. Five clones (including #XE501) encoding a fragment of
Caspase-1 (L89-G403) were isolated. The remaining five clones were
apparent false positives.
Flow Chart for cDNA Library Screening Using Reporter Gene Strategy
Based on Cleavable Transcription Factor.
[0101] The example shown in FIG. 15 is S3/S7 substrate. Yeast
strain EGY48 containing 6op-LEU2 and 2op-lacZ reporter genes and
the DEVD containing cleavable transcription factor (expressed from
TEF-Fas-d-S3-TA) was transformed with a HEK293 cell cDNA library
(random-primed, PGAL1 promoter, HAtagged) (left). Independent
colonies (.about.1.0.times.106) appeared on growth plates within 48
hours. Cells were collected, pooled, and a portion (5.0.times.106)
was seeded on the leucinedeficient selection plates containing
X-gal. Blue-colored colonies (n=21) appeared within a week, of
which 3 clones (including #ZB331) encoded full-length Caspase-3,
while the rest were false positives. In a similar experiment
(right), another HEK293 cDNA library (oligo-dT-primed, PADH
promoter) (1.8.times.106 initial colonies) was screened, resulting
in 9 positive clones (including #X8312) encoding full-length
Caspase-7. The remaining five clones were false positives. Both
cloned Caspases are known to cleave DEVD tetrapeptide [Thornberry,
N. A, et. al., J. Biol. Chem. 272, 17907-17911 (1997)].
Use of One-component Yeast-based Caspase Activity Assay for cDNA
Library Screening.
[0102] FIG. 16 consisting of 16A and 16B demonstrates the results
of a one-componsnt yeast-based Caspase activity assay for cDNA
library screening. (A) The plasmids containing library cDNAs from
FIG. 14 were recovered and re-transformed into yeast cells
containing 6op-LEU2 and 2op-lacZ reporter genes with either
cleavable (WEHD-containing) (expressed from TEF-Fas-d-S1-TA) or
non-cleavable (WEHG-containing) transcription factor substrate
(expressed from TEF-Fas-d-G1-TA) to confirm whether they cleave S1
specifically. As controls, yeast were transformed with a plasmid
encoding Caspase-1 ("Positive" control) or the empty vector
("Negative" control). Assays were performed in duplicate, with
cells grown on selection plates for 4 days. Note that the cDNA
library clones supported lacZ reporter gene activation only when
co-expressed with the S1 cleavable substrate. (B) The cDNA library
plasmids from FIG. S3 were recovered and used to re-transform the
yeast strains containing either the same cleavable DEVD-containing
or DEVG noncleavable transcription factor, expressed from
TEF-Fas-d-S3-TA ("S3" substrate) and TEF-Fas-d-G3-TA ("G3"
substrate), respectively. Assays were performed in duplicate and
cells grown on selection plates for 4 days. Note that the two cDNA
library clones activated the lacZ reporter gene only when
co-expressed with cleavable S3 (DEVDcontaining) substrate. As
controls, yeast cells were transformed with plasmids encoding
Caspase-3 ("Positive" control) or the empty vector ("Negative"
control).
Validation of Yeast-based Assays for Effector Caspase
Activators--Two and Three-component Systems.
[0103] As shown in FIG. 17 consisting of 17A, and 17B yeast EGY191
strain containing 2op-LEU2/2op-lacZ or EGY48 strain containing
6op-LEU212op-lacZ were employed for developing assays for
activators of downstream effector Caspases (e.g. Caspases-3 and
-7). Yeast transformants were plated on leucine-deficient medium
containing X-gal. Yeast were transformed with plasmids encoding
membrane tethered transcription factor substrate with either
DEVD-containing cleavable (S3) or DEVG-containing non-cleavable
(G3) linkers, and with plasmids encoding pro-Caspase-3 (C3) or
pro-Caspase-7 (C7) or the corresponding empty vector (-). (Note
that the optimal tetrapeptide sequence for both Caspase-3 and
Caspase-7 has previously been reported to be DEVD). Yeast
transformants were as follows: (A) S3 Substrate/No Caspase:
EGY191-2op-LEU2/2oplacZ/D.DELTA.TEF3-Fas-d-S3 (DEVD)-TA; S3
Substrate/Caspase-3:
EGY191-2op-LEU2/2oplacZ/D.DELTA.TEF3-Fas-d-S3/7(DEVD)-TA/D.DELTA.CYC2-HA--
Caspase3; G3 Substrate/Caspase-3:
EGY191-2op-LEU2/2op-lacZ/D.DELTA.TEF3-Fas-d-G3/7(DEVG)-TA/D.DELTA.CYC2-Ca-
spase-3; and (B) S3 Substrate/No Caspase:
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S3(DEVD)-TA; S3
Substrate/Caspase-7:
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S3(DEVD)-TA/CYC-Caspase-7; G3
Substrate/Caspase-7: EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G3
(DEVG)-TA/CYC-Caspase7. These yeast transformants were then
subsequently transformed with plasmids expressing a small amount of
Caspase-9 (driven from the CYC promoter) or plasmids expressing
large amounts of wild-type (WT) or catalyticallydefective
(C287.fwdarw.A287) Caspase-9 (driven from the TEF promoter), with
or without an active form (gain of function mutant not requiring
cytochrome c for activation) of Apaf-1 (driven by TEF-promoter) or
the corresponding empty vectors (-) (Haraguchi M, Torii S,
Matsuzawa S, et al. J Exp Med 2000; 191: 1709-20).
[0104] Note that the large amount of Caspase-9 (expressed from TEF
promoter) activated the cleavable S3(DEVD) substrate when
co-expressed with pro-Caspase-3 or pro-Caspase-7, but not in their
absence, thus constituting a 2-component system. No lacZ reporter
gene activity was detected when the non-cleavable substrate (DEVG)
was employed (G3). In contrast, expressing a small amount of
Caspase-9 (from the CYC promoter) or the catalytically-defective
Caspase9 (C287.fwdarw.A287) did not activate Caspase-3 or
Caspase-7. However, co-expressing active Apaf-1* with a small
amount of pro-Caspase-9 activated the lacZ reporter gene in yeast
expressing pro-Caspase-3 or -7 (but not in the absence of these
downstream effector Caspases), thus constituting a 3-component
system.
Validation of Yeast-based Assays for Initiator Caspase Activators:
Two Component Systems.
[0105] As shown in FIG. 18 consisting of 18A, 18B, 18C and 18D
yeast EGY191 strain containing 2op-LEU2/2op-lacZ or EGY48 strain
containing 6op-LEU212op-lacZ were employed for developing
2-component assays for activators of upstream initiator Caspases
(e.g. Caspases-1, 2, 8, 9, 10). Two independent clones of yeast
transformants were plated on leucine-deficient medium containing
X-gal. Yeast were transformed with plasmids encoding various
membrane tethered transcription factor substrates containing (A)
WEHD ("S1"), (B) DEHD ("S2"), (C) LEHD ("S9") or (D) LETD ("S8")
cleavable linkers or their corresponding noncleavable glycine
mutants ("G1", "G2", "G9", "G8"). The yeast were also transformed
with plasmids encoding wild-type (WT) or catalytically inactive
mutants of proforms various initiator Caspases expressed from
relatively weak promoters (e.g., CYC1; .DELTA.TEF3), including (A)
pro-Caspase-1, (B) pro-Caspase-2, (C) pro-Caspase-9, and (D)
pro-Caspase-10. (Note that the optimal betrapeptide sequences are
the same for Caspase-8 and 10). These yeast were transformed with
empty vectors (-) or plasmids encoding upstream activators of the
Caspases, including (A) Asc, (B) RAIDD, (C) Apaf-1*, and (D) FADD,
expressed from strong promoters (either GPD or TEF).
[0106] Note that the lacZ reporter gene was activated only when the
combination of an initiator Caspase and upstream activator was
co-expressed, along with a cleavable substrate.
[0107] Transformants. The transformed yeast cell clones are: (A)
EGY191-2op-LEU2/2oplacZ/TEF-Fas-d-S1
(WEHD)-TA/.DELTA.TEF1-Caspase1-FLAG (S1, C1(WT)),
EGY191-2op-LEU2/2op-lacZ/TEF-Fas-d-G1
(WEHG)-TA/.DELTA.TEF1-Caspase1-FLAG (G1, C1(WT)), or
EGY191-2op-LEU2/2op-lacZ/TEF-Fas-d-S1(WEHD)-TA/.DELTA.TEF1-Caspase1
(C285.fwdarw.G285)-FLAG, (S1, C1(C285.fwdarw.G285)), were
transformed with the plasmids encoding the activator Asc, or the
empty vector (-); (B)
EGY48-6op-LEU2/2op-lacZ/.DELTA.TEF1-Fas-d-S2(DEHD)-TA/.DELTA.GPD1-HA--
Caspase2-FLAG(S2, C2(WT)),
EGY48-6op-LEU2/2op-lacZ/.DELTA.TEF1-Fasd-G2(DEHG)-TA/.DELTA.GPD1-HA-Caspa-
se2-FLAG (G2, C2(WT)), or
EGY48-6op-LEU2/2op-lacZ/.DELTA.TEF1-Fas-d-S2(DEHD)-TA/.DELTA.GPD1-HA-Casp-
ase2 (C320.fwdarw.A320)-FLAG (S2, C2(C320.fwdarw.A320)), were
transformed with plasmids encoding the activator RAIDD, or the
empty vector (-); (C)
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S9(LEHD)-TA/TEF-HACaspase9 (S9,
C9(WT)),
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G9(LEHG)-TA/TEF-HA-Caspase9 (G9,
C9(WT)), or
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S9(LEHD)-TA/TEF-HA-Caspase9(C287.fwdarw-
.A287) (S9, C9(C287.fwdarw.A287)), were transformed with plasmids
encoding an active form of Apaf-1 (Apaf*), or the empty vector (-);
(D)
EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/ADH-Caspase10-FLAG
(S8, C10(WT)),
EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-G8(LETG)-TA/ADH-Caspase10-FLA- G
(G8, C10(WT)), or
EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/ADHCaspase10(C358.fwdarw.A3-
58)-FLAG (C10(C358.fwdarw.A358)), were transformed with the
plasmids encoding the activator FADD, or the empty vector (-). The
large amount of FADD is enough to activate Caspases-10 by
itself.
Specificity of Upstream Activators of Initiator Caspases--Tested by
2-component Systems.
[0108] As shown in FIG. 19 yeast transformants were prepared and
tested as described in FIGS. 17 and 18, except a matrix of plasmid
combinations was prepared to evaluate the specificity of upstream
activators. The substrates and Caspases tested are indicated across
the top, while the activators are indicated along the side.
[0109] Note that results obtained were as predicted, with (1)
Apaf-1* activating pro-Caspase-9, but not other initiator Caspases;
(2) RAIDD activating pro-Caspase-2, but not other Caspases; (3)
FADD activating pro-Caspases-8 and 10, but not other Caspases, and
(4) Asc activating pro-Caspase-1 and 8. Note that while Asc
contains a CARD that pairs with a complementary CARD in
pro-Caspase-1 and would not be necessarily predicted to activate
the DED-containing protease Caspase-8, it has previously been
reported that Asc is an activator of Caspase-8 (Hasegawa M. et. al.
J Biol Chem 280: 15122-30 (2005); Masumoto, J. et. al., Biochem.
Biophys. Res. Commun 303: 69-73 (2003).
[0110] Transformants: Transformed yeast clones were as follows:
S1,C1:EGY191-2op-LEU2/2op-lacZ/TEF-Fas-d-S1
(WEHD)-TA/P.DELTA.TEF1-Caspase1-FLAG; S2, C2:
EGY48-6op-LEU2/2op-lacZ/P.DELTA.TEF1-Fas-d-S2(DEHD)-TA/P.DELTA.GPD1-HA-Ca-
spase2-FLAG; S8, C8:
EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8/10(LETD)-TA/CYC1-Caspase8-HA;
S9, C9:
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S9(LEHD)-TA/TEF-HA-Caspase-9; S8,
C10:
EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8/10(LETD)-TA/ADH-Caspase10-FLAG.
These cells were transformed with the plasmids encoding the
activators (Asc, RAIDD, FADD, and Apaf*). For controls (-), the
"empty" version of the plasmids were introduced.
Validation of 3-component Yeast-based Caspase Assay Reconstituting
DISC.
[0111] As shown in FIG. 20 consisting of 20A and 20B yeast
transformants were prepared to assess the performance of the
Fas/FADD/Caspase-8/10 three-component systems. Two independent
clones of each transformant were plated on leucine-deficient medium
containing X-gal. Substrates included LETD-containing cleavable
(S8) and LETG-containing non-cleavable (G8) transcription factors,
while Caspase expression plasmids included WT pro-Caspases-8(A) and
-10 or a catalytic mutant of Caspase-10. (B) FADD was expressed at
low levels from a CYC1 promoter. Note that the lacZ reporter gene
was activated only when the combination of Fas, FADD, and either WT
pro-Caspase-8 or -10 was co-expressed and only when a cleavable
substrate (S8) was employed. (Note that the optimal tetrapeptide
cleavage sequence is the same for Caspases-8 and -10).
[0112] Transformants: (A) Yeast transformants included
EGY48-6op-LEU2/2op-lacZ/TEFFas-d-S8 (LETD)-TA/CYC1-Caspase-8-HA
(S8, C8)
EGY48-6op-LEU2/2op-lacZ/TEFFas-d-G8(LETG)-TA/CYC1-Caspase8-HA (G8,
C8), without (-) or with Fas, and without (-) or with FADD, which
were expressed from either ADH and .DELTA.ADH or CYC1 promoters,
respectively, to achieve high expression of Fas and low expression
of FADD. (B) Yeast transformants included:
EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10-FLAG
(S8, C10);
EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-G8(LETG)-TA/CYC1-Caspase10-FLAG
(G8, C10); and EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8
(LETD)-TA/CYC1-Caspase10(C358.fwdarw.A358)-FLAG (S8,
C10(C358.fwdarw.A358)) each without (-) or with Fas, and without
(-) or with FADD-expressing vector or the corresponding empty
vectors.
Flow Chart for cDNA Library Screening Using 3 Component
System--Application to Death Receptor Cloning--Example Screening
Strategy #1.
[0113] FIG. 21 demonstrates an exemplary screening screening
strategy is essentially the same as outlined in FIGS. 6 and 7. The
yeast transformant
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S8(LETD)-TA/CYC1-Caspase8-HA/.DELTA.GDH-
1 -FADD was transformed with a HepG2 cell cDNA library (oligo
dT-primed, PADH promoter). Independent colonies (1.7.times.106)
appeared on growth plates within 48 hours. Cells were collected,
pooled, and a portion (2.4.times.107) was seeded on the
leucine-deficient selection plates containing X-gal. Blue-colored
colonies (n=150) appeared within a week, 13 of which encoded DR4,
(#XA514), and 15 clones encoded DR5 variant 2 (#ZA512), while the
rest were false positives. Screening another cDNA library from
HepG2 cells (random-primed, PADH promoter) yielded eight DR5
(#XD108) clones.
Flow Chart for cDNA Library Screening Using 3 Component System to
Clone Death Receptors--Example Screening Strategy #2.
[0114] FIG. 22 demonstrates an exemplary screening he screening
strategy is essentially the same as outlined in FIGS. 6 and 7. The
yeast transformant
EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S8(LETD)-TA/CYC1-Caspase8-HA/.DELTA.PDH-
1-FADD was transformed with a human liver cDNA library
(random-primed, PADH promoter) and a HeLa cell cDNA library
(randomprimed, PADH promoter).
Flow Chart for cDNA Library Screening Using 3-component System to
to Clone Death Receptors--Example Screening Strategy #3.
[0115] FIG. 23 demonstrates an exemplary screening is essentially
the same as outlined in FIGS. 6 and 7. The yeast transformant
EGY48-6op-LEU2/2op-lacZ/TEFFas-d-S8(LETD)-TA/CYC1-Caspase8-HA/.DELTA.PDH1-
-FADD was transformed with a HEK293T cell cDNA library
(oligo-dT-primed, PADH promoter) and a HEK293T cell cDNA library
(random-primed, PADH promoter, HA-tagged).
Schematic Representation of 3-component System Used for Cloning
Adapter Protein that Links Fas to Pro-Caspase-10.
[0116] FIG. 24 consisting of 24A and 24B represents a 3-component
system for cloning an adapter protein that links Fas to
pro-Caspase-10. (A) The zymogen pro-Caspase-10 was expressed (from
CYC1-promoter) with substrate containing the LETD-containing
cleavage element S8 (from plasmid
p413-TEF-Fas-d-S8-TA/CYC1-Caspase-10-FLAG) in the yeast EGY48
expressing 6op-LEU2. Fas was also expressed without activating the
Caspase-8 from plasmid p426-2op-lacZ/ADH-Fas. (B) Addition of FADD
activates Caspase-8, releasing the transcription factor.
Validation of Adapter Protein Cloning System for
Fas/FADD/Caspase-8/10: Reconstituted DISC.
[0117] As shown in FIG. 25 consisting of 25A and 25B yeast cell
transformants were prepared to assess the performance of the
Fas/FADD/Caspase-8 or Fas/FADD/Caspase-10 three-component systems.
Two independent clones of each transformant were plated on
leucinedeficient medium containing X-gal and grown for 4 days.
Substrates included LETD17 containing cleavable (S8) and
LETG-containing non-cleavable (G8) transcription factors, while
Caspase expression plasmids included wild-type (WT) pro-Caspases-8
(A) and -10 or a catalytic mutant of Caspase-10 (C358.fwdarw.A358)
(B). Note that the lacZ reporter gene was activated only when the
combination of Fas, FADD, and either WT pro-Caspase-8 or -10 was
co-expressed and only when a cleavable substrate (S8) was
employed.
[0118] Transformants: (A) Yeast cell transformants included:
EGY48-6op-LEU2/2op-lacZ/TEFFas-d-S8 (LETD)-TA/CYC1-Caspase8-HA (S8,
C8) and EGY48-6op-LEU2/2oplacZ/TEF-Fas-d-G8
(LETG)-TA/CYC1-Caspase8-HA (G8, C8), with empty vector (-) or with
plasmids encoding FADD (expressed from CYC1 promoter) or Fas
(expressed from ADH promoter). (B) Yeast cell transformants
included
EGY48-6op-LEU2/2oplacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10-FLAG
(S8, C10), EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-G8
(LETG)-TA/CYC1-Caspase10-FLAG (G8, C10), and
EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8
(LETD)-TA/CYC1-Caspase10(C358.fwdarw.A358)-FLAG (S8,
C10(C358.fwdarw.A358)), each with empty vector (-) or with plasmids
encoding FADD or Fas as above.
Flow Chart for cDNA Library Screening Using 3 Component System to
Clone Adapters.
[0119] As shown in FIG. 26 the yeast transformant
EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S
8(LETD)-TA/ADH-Fas/CYC1-Caspase10-FLAG was transformed with a
HEK293 cell cDNA library (random-primed, PADH promoter, HA-tagged).
Independent colonies (.about.2.2.times.106) appeared on growth
plates within 48 hours. Cells were collected, pooled, and a portion
(3.2.times.107) was seeded on leucine-deficient selection plates
containing X-gal. Blue-colored colonies (n=200) appeared within a
week, five of which encoded fulllength Caspase-2 (#XA227), five
encoded a fragment of Caspase-2 (#ZA214), five encoded a fragment
of Caspase-8 (#XA221), and 24 encoded full-length FADD (#XA212),
while the rest were apparent false positives.
Examples of cDNA Cloning Results.
[0120] As shown in FIG. 27 consisting of 27A, 27B and 27C the
clones that activated the reporter genes were characterized by
recovery of cDNA library plasmids and retransformation into yeast
expressing Fas and pro-Caspase-10(A), pro-Caspase-10 without Fas
(B), or neither (C). Among the positive clones were a full-length
Caspase-2 (#XA227), a fragment of Caspase-2 (#ZA214, V130-L312), a
fragment of Caspase-8 (#XA221, M1-K438), and full-length FADD
(#XA212). Assays were performed in duplicate, growing cells for 4
days on plates.
Optimization of Signal:noise Ratio in Microtiter Plates: Cell
Density.
[0121] As shown in FIG. 28 consisting of 28A, 28B, and 28D
demonstrated that EGY48 cells containing the 3-component
Fas/FADD/Caspase-8 system and Caspase-8-cleavable reporter were
seeded at 2.35.times.104/well (A), 2.times.105/well (B), or
3.76.times.105/well (C) in 384 well plates to compare cell
densities. Cells were cultured at 30.degree. C. The activity of
.beta.-galactosidase was measured after 72 hrs at various times
after initiating cultures (mean+std dev; n=3), for cells grown
without (white circles) or with (black circles and black squares)
X-gal and grown in the absence (circles) or presence (black
squares) of 100 .mu.M zVAD-fmk Caspase inhibitor.
Optimization of Signal:noise Ratio in Microtiter Plates: Time and
X-gal Concentration.
[0122] FIG. 29 consisting of 29A, and 29B demonstrates in (A) EGY48
cells containing the 3-component Fas/FADD/Caspase-8 system and
Caspase-8-cleavable reporter were grown at 2.times.105 cells/well
in 384 well plates, comparing .beta.-galactosidase activity at
various times (mean.+-.std dev; n=3), for cells grown in the
absence (black squares) or presence (circles) of X-gal and in the
absence (circles) or presence (squares) of 100 .mu.M zVAD-fmk
Caspase inhibitor. (B) EGY48 cells containing the 3-component
NLRP1.DELTA.LRR/Asc/Caspase-1 system and Caspase-1-cleavable
reporter were grown at 2.times.105 cells/well in 384 well plates,
comparing X-gal concentrations. The activity of
.beta.-galactosidase was measured after 72 hrs culture (mean.+-.std
dev; n=3) for cells grown in the absence (red circles) or presence
(all others) X-gal. Various concentration of X-gal were compared,
where 1.times.=50 ug/mL final concentration.
Plasmids for Co-expression of Caspases and Substrate Cleavable
Transcription Factors in Yeast.
[0123] As shown in FIG. 30 consisting of 30A, 30B, 30C and 30D the
plasmid p413 was used as the backbone for these constructions,
containing CEN/ARS centromeric origin for low-copy episomal
replication in yeast (S. cerevisiae) and HIS3 gene for selection in
his3 yeast strains. (A) The plasmid
p413-TEF-Fas-d-S1-TA/.DELTA.TEF3-Caspase-1-FLAG, contains two
additional transcriptional units, where expression of the
Fas-LexA/B42 membrane tethered transcription factor with WEHD
linker (Caspase-1/4/5 cleavable) is driven by the TEF promoter and
expression of pro-Caspase-1 with C-terminal FLAG tag is driven by
an attenuated TEF3 promoter (.DELTA.TEF3). (B) The plasmid
p413-TEF-Fas-d-S8-TA/CYC1-Caspase-8-HA contains two additional
transcriptional units, where expression of the Fas-LexA/1B42
membrane tethered transcription factor with LETD linker
(Caspase-8/10 cleavable) is driven by the TEF promoter and
expression of pro-Caspase-8 with C terminal HA tag is driven by
CYC1 promoter. (C) The plasmid
p413-GDP-Fas-dS8-TA/CYC1-Caspase-10-FLAG, similarly contains two
additional transcriptional units, where expression of the same
Caspase-8/10 cleavable Fas-LexA/B42 substrate as above is driven by
GDP promoter and where expression of pro-Caspase-8 with C-terminal
FLAG tag is driven by CYC1 promoter. Transcriptional termination
elements from the CYC1 and ADH genes were employed as
illustrated.
Plasmids for Expression of Upstream Activators of Caspases and lacZ
Reporter Gene in Yeast.
[0124] As shown in FIG. 31 consisting of 31A, 31B, 31C and 31D the
plasmid p426 was used as the backbone for these constructions,
containing 2.mu. plasmid origin for high-copy episomal replication
in yeast and URA3 gene for selection in ura3 yeast strains. (A) The
plasmid p426-2op-lacZ contains a lacZ gene driven by a minimal
promoter containing two tandem copies of binding sites for the LexA
transcription factor, followed by ADH gene termination element. (B)
Plasmid p426-2op-lacZ/CYC1-HA-Asc contains an additional
transcriptional unit, where expression of human cDNA encoding
N-terminal HA-tagged Asc protein is driven by a CYC1 promoter, and
followed by ADH gene termination element. (C) In plasmid
p426-2op-lacZ/CYC1-FADD, expression of a human FADD cDNA is driven
by CYC1 promoter and followed by ADH termination element. (D)
Plasmid p426-2op-lacZ/ADH-Fas contains a human Fas(CD95) cDNA
driven by ADH promoter, and followed by CYC1 gene termination
element.
Yeast Expression Plasmids for Functional Screening of cDNA
Libraries and Expression of Upstream Activators of Caspases.
[0125] As shown in FIG. 32 consisting of 32A, 32B, and 32C the
plasmid p424 was used as the backbone for these constructions,
containing 2.mu. plasmid origin for high-copy episomal replication
in trp1 yeast and TRP1 gene for selection in .DELTA.TRP1 yeast
strains (S. cerevisiae). (A) The plasmid p426-GAL1-cDNAs contains
human cDNAs directionally cloned downstream of a GAL1 promoter,
followed by CYC1 gene termination element. (B) Plasmid
p424-CYC1-HA-Asc contains a human cDNA encoding N-terminal
HA-tagged Asc protein cloned downstream of a CYC1 promoter and
followed by CYC1 gene termination element. (C) In plasmid
p424-CYC1-HAAsc/TEF-NLRP1.DELTA.LRR, an additional transcriptional
unit was added to p424-CYC1-HAAsc above, where expression of a
human cDNA encoding a gain-of-function NLRP1 mutant lacking LRRs
(Faustin, B, et al. Molecular Cell 25; 713, 2007) is driven by a
TEF promoter and followed by ADH gene termination element.
Experimental Designs & Methods Used
[0126] Chemical Library Screens of Multi-component Yeast-based
Protease Assay Systems were Performed to Define Hit-rates and Test
Reliability.
[0127] Chemical library screens to validate the performance of the
assays were completed. The screens were conducted such that all
assay components were added in automated fashion using integrated
robotic liquid handling systems, moving the plates initially into
carousels that hold 180 plates at room temperature, and then
manually applying seals (breathable sealing film from Axygen
Scientific) to reduce evaporation, and moving the bar-coded plates
to a 30.degree. C. incubator for the required time (generally
culturing for 2-4 days or less). Each assay plate contained a row
of positive (min) and a row of negative (max) controls that did not
receive compounds but that received DMSO in volumes equivalent to
the amount of DMSO in which compounds will be supplied. At the
conclusion of the 72 hrs incubation, plates were robotically
delivered to one of the integrated multi-purpose plate readers for
reading at OD620 nm. The programmable robotic workstations
sequenced the additions of reagents, minimizing variations in
incubation times. Data from primary screens were uploaded directly
from plate readers into computers with customized Microsoft excel
software, set up to calculate Z' factor for each plate, and with
hit determinations set at 50% of the mean value for the negative
control values.
[0128] A screening of a LOPAC was performed at several different
concentrations (typically 20, 10, and 5 .mu.M) to compare the hit
rates, and to empirically determine an acceptable concentration for
conducting large-scale library screens. The empirically adjusted
compound screening concentrations allowed for the employment of the
highest screening concentration tolerable without causing artifacts
or non-specific effects. A hit rate of 0.1-0.5% was achieved which
was consistent with the general expectations for the screening
results. Before undertaking this study, it was empirically
determined what the effects of DMSO on assay performance would be,
through pilot experiments where increasing concentrations of DMSO
(from 1-10% volume) were added to the positive and negative
controls and the impact on assay signal and stability was
determined. Second, a progression was made to larger libraries
(eg., 50K Chembridge library), it was determined which factors
improve the stability of the positive and negative controls from
plate to plate, assessing the assay performance as time is extended
from minutes to hours, and calculating Z' for each plate as the
quality of the assay's performance is assessed in true screening
mode.
[0129] For HTS assays the .beta.-galactosidase produced by yeast
carrying the caspase-cleavable reporter proteins was measured, by
assaying the calorimetric product (OD620 nm) derived from X-gal
substrate in 384 well plates, as a measure of the lac Z reporter
gene activity.
[0130] In summary disclosed are HTS systems for intracellular
proteases, using Caspases as a prototype. For this purpose,
yeast-based cellular systems that permit facile expression of
proteases and protease-activating proteins in combinations that
reconstitute entire mammalian pathways in these simple eukaryotes
were devised. Among the assay methods integrated into the yeast
system are cleavable reporter gene activators, in which
protease-mediated cleavage activates a transcription factor. In
summary we disclose:
1. Multi-component systems that reconstitute mammalian protease
activation pathways in yeast. 2. Optimized systems with adjusted
expression levels, reporter gene sensitivity, and other parameters
to achieve satisfactory signal:noise results and mechanisms for
optimizing future systems. 3. Defined the key variables that
require optimization for achieving HTS quality assay performance.
4. Performed chemical library screens of multi-component
yeast-based protease assay systems to define hit-rates and test
reliability.
Plasmid Constructions.
Vectors Encoding Caspase Cleavable Transcription Factors:
[0131] The reporter, Fas-d-S1-TA for Caspase-1 and related
proteases (called "S1"), was generated by using PCR and standard
recombinant DNA techniques. This protein consists of, from N to C
termini, amino acids M1-L224 of a type 1 transmembrane protein,
human Fas 34 in which Fas is devoid of the cytosolic death domain
(Fas-d), a linker containing the sequence GWEHDG between a Xhol and
EagI site, and finally a transcriptional activator (TA) containing
the LexA DNA binding domain and the B42 activation domain, taken
from plasmids pRS305(.DELTA.wbpl-Cub-PLV) 35 and pJG4-5
(Invitrogen) with PCR. Other reporters were made by substituting
the linker with oligonucleotides designed to encode in-frame the
sequences GWEHGG ("G1"), GDEHDG ("S2"), GDEHGG ("G2"), GDEVDG
("S3"), GDEVGG ("G3"), GTEVDG ("S6"), GTEVGG ("G6"), GLETDG ("S8"),
GLETGG ("G8"), GLEHDG ("S9"), or GLEHGG ("G9"), after digestion
with Xhol and EagI.
Plasmids for Expressing Caspases and Their Upstream Activators:
[0132] Plasmids for expression of Caspases in yeast were derived
from pRS series vectors 36-38 and Pesc series vectors (Stratagene
[Agilent]). Expression levels were adjusted by using constitutive
promoters of different strengths (CYC1, ADH, TEF, and GPD) 39 or
using the inducible GAL1 promoter, in conjunction with different
strength reporter genes carrying variable numbers of lexA operators
6, and using plasmids with different replication origins for low or
high copy number replication (2u and CEN/ARS). To further control
expression levels, several attenuated forms of the promoters were
made by PCR-assisted deletional mutagenesis. Using a standard
indicator gene, the approximate relative strength of the promoters
was determined to be:
.DELTA.CYC4<.DELTA.CYC2<CYC1<.DELTA.GPD2<.DELTA.GPD1<.DELT-
A.ADH1<ADH<.DELTA.TEF3<.DELTA.TEF2<.DELTA.TEF1<TEF.
However, the relative locations of the promoters in complex
plasmids somewhat affects their strength, especially when two or
three genes are contained in one plasmid. In this regard, two or
three genes within one plasmid were sometimes co-expressed by
placing the genes flanked by the above promoters and inserting
transcription termination elements between them, because the
selectable marker genes available are limited (URA3, HIS3, TRP1,
and LEU2). In addition, many of the expressed Caspases and upstream
activators were cloned with N-terminal HA or C-terminal HA or FLAG
epitope tags for convenience of detection of the protein products
by immunoblotting. Examples of complex plasmids are (a)
p426-2op-lacZ/CYC1-FADD, which consists of 2.mu. origin, URA3
marker, lacZ gene under the control of two lexA operators, and a
FADD gene with expression driven by a short form of the ADH
promoter (.DELTA.ADH1), as in FIG. 31C, and (b)
p413-TEF-Fas-d-S8-TA/CYC1-Caspase8-HA, which consists of CEN/ARS
origin, HIS3 marker, Fas-d-S8-TA substrate driven by TEF promoter,
and a C-terminally HA-tagged pro-Caspase-8 cDNA under control of
the CYC1 promoter, as in FIG. 30C.
Complex Plasmids Constructions.
[0133] Diagrams of complex plasmids containing 3 or more genes are
provided in FIGS. 30-32. The cDNAs encoding human Caspases, Adapter
proteins, Death Receptors, and NLRs, in wild-type and mutant
versions have been described previously J- M. Bruey, et al., Nature
Cell Biology 2, 645 (2000). M. Krajewska, et al., Exp Neurol 189
(2), 261 (2004). H. Marusawa, et al., EMBO J 22, 2729 (2003). T.
Miyazaki, et al., Nature Immunol 3, 4 (2002). A. D. Schimmer, et
al., Cancer Cell 5, 25 (2004).
Construction of Expression cDNA Libraries.
[0134] Oligo(dT)-primed or random heptamerprimed cDNA libraries
were made in modified p424-GAL1, p424-GAL1-HA, p424-ADH, or
p424-ADH-HA plasmids (carrying a TRP1 marker) using mRNAs derived
from HEK 293 cells, HepG2 cells, HeLa cells, human liver, or human
placenta, as in FIG. 32A. The mRNAs of HepG2 cells, HeLa cells,
human liver, and human placenta were purchased from Ambion. The
p424-GAL1 was modified to create 5'XhoI and 3'NotI sites downstream
of a GAL1 promoter for insertion of the cDNAs. The p424-GAL1-HA has
a HA-tag between the GAL1 promoter and the 5'Xhol site. The
p424-ADH and p424-ADH-HA plasmids were modified with 5'XhoI and
3'SfiI sites downstream of the ADH promoter. To construct
directional libraries, the first-strand cDNA syntheses were
initiated with a NotI-oligo(dT) primer adaptor or a NotI-random
hexamer primer adaptor for the p424-GAL1 and p424-GAL1-HA, and with
a Sfiloligo(dT) primer adaptor or a SfiI-random hexamer primer
adaptor for the p424-ADH and p424-ADH-HA, according to the
manufacturer's instructions (Invitrogen). SalI adapters were
ligated to the resultant double-strand cDNAs prior to digestion
with NotI or SfiI. The cDNAs with SalI-NotI termini or with
SalI-SfiI termini were ligated into the plasmid cloning vectors
(XhoI-NotI-cut or XhoI-SfiI-cut, respectively). The number of
initial transformants ranged from 1.3.times.106 to 1.3.times.107. A
human B cell cDNA library was purchased from ATCC. The
oligo(dT)-primed cDNAs were inserted into the XhoI cloning site,
downstream of a GAL1 promoter.
Reporter Gene Assays.
[0135] Plasmids were introduced into yeast by lithium acetate
transformation. The yeast strain EGY48, which carries 6 lexA
operators upstream of LEU2 gene (6op-LEU2), was transformed with
pJK103 6, which carries two lexA operators upstream of lacZ gene
(p426-2op-lacZ), and subsequently with reporter plasmids
(p413-TEF-Fas-d-S1-TA, p413-TEF-Fas-d-G1-TA, p413-TEF-Fas-d-S2-TA,
p413-TEF-Fas-d-S3-TA, p413-TEF-Fas-d-S6-TA, p413-TEF-Fas-d-S8-TA,
or p413-TEF-Fas-d-S9-TA). Caspase (all full-length zymogen
proforms) expression plasmids (p424-ADH-Caspasel-FLAG,
p424-ADH-HA-Caspase2, p424-ADH-Caspase3, p424-TEF-Caspase4,
p424-ADH-Caspase5, p424-TEF-HA-Caspase6, p424-ADH-Caspase7, and
p424-ADH-HA-Caspase9-FLAG) or empty vector (p424-ADH), were
introduced into these backgrounds. As for assays with Caspase-8 and
-10, small amounts of pro-Caspase-8 and -10 were expressed with a
large amount of FADD by transforming yeast with the dual gene
plasmids p424-CYC1-Caspase8-HA/TEF-HA-FADD and
p424-CYC1-Caspase10-FLAG/TEF-HA-FADD, because expression of large
amounts of human Caspase-8 or Caspase-10 significantly inhibited
the cell growth. The transformants (two independent colonies for
each transformation) were streaked on growth plates (minimum
synthetic dropout (SD) medium containing 2% glucose and 50 .mu.g/ml
leucine) or on selection plates (SD medium containing 1% galactose,
0.2% raffinose, BU salts, and 80 .mu.g/ml X-gal). Yeast growth and
blue color development were monitored for four to six days at
30.degree. C.
Reporter Gene Assays in Liquid Media Using 384-well Plates.
[0136] Assays were performed in a total volume of 40 .eta.l in
triplicate. First, 20 .mu.l of liquid selection media containing
X-gal and a series of concentrations of reagents such as z-VAD was
dispensed into each well of 384-well plates. Next, confluent yeast
cells expressing various Caspases and cleavable substrates were
collected, washed with water, and suspended in selection media of
the same volume as the culture media. The yeast suspension was
diluted to 1:5-10 (v:v) with the selection media, and 20 .mu.l
aliquots were added to the 384-well plates. Absorbance at 620 nm
was measured 2-3 days after culture at 30.degree. C.
HTS Assays.
[0137] For the Fas/FADD/Caspase-8 and the NLRP1/Asc/Caspase-1
assays, EGY48 yeast containing the desired plasmids were streaked
onto SD plates (6.8 g Yeast Nitrogen Base w/o amino acids, 20 mg
arginine, 50 mg threonine, 30 mg isoleucine, 60 mg phenylalanine,
20 mg valine) containing agar (1.7%), supplemented with 2%
.alpha.-D-glucose and 50 .mu.g/mL leucine. The plates were
incubated at 30.degree. C. for 48 hrs and a colony was picked and
transferred to a 14 ml polypropylene tube containing 2 ml of SD
media broth supplemented with .alpha.-D-glucose and leucine as
above and grown at 30.degree. C. for 16-24 hrs with shaking. Then,
1 ml of the overnight culture was transferred into 20 mls of growth
media in a 500 ml flask and shaken at 30.degree. C. for 16-24 hrs.
The cells were collected by centrifugation at 1000.times.g for 5
minutes at room temperature, and washed with 20 mls of sterile
water, then resuspended in 20 mls of SD broth supplemented with 1%
galactose and 0.2% raffinose. The HTS Assay was performed at a
final compound concentration of 10 .mu.M (1% DMSO), with cell
densities of 2.times.105 cells/ml (2.times.105 cells) in a volume
of 40 .mu.l. The assay was assembled by the addition of 4 .mu.l of
.apprxeq.100 .mu.M compounds (final concentration of 10 .mu.M) in
10% DMSO (1% final DMSO) to clear polystyrene 384-well microplates
using a Beckman-Coulter Biomek FX, then 18 .mu.l of cell suspension
(1.1.times.107 cells/ml) (2.times.105 cells/well) and 18 .mu.l of
X-gal suspended in Selection Media (for a final concentration of
400 .mu.g/mL) were added to the wells using a Matrix WellMate bulk
reagents dispenser. Remaining solutions were added by using the
WellMate from Matrix Technologies Corp. Controls were included with
each plate, corresponding to cells treated with DMSO (without
compounds) and cells cultured with and without X-gal. The plates
were sealed with breathable sealing film (from Axygen Scientific)
to reduce evaporation and transferred to 30.degree. C. incubators.
After 48 hours, the plates were brought to room temperature, the
breathable film removed and replaced with the transparent polyester
tape seal, and the plates were mixed, pelleted briefly and read
using a Beckman DTX 880, recording absorbance at 620 nm.
Yeast-based Counter-screen Assays.
[0138] EGY48 yeast inducibly (GAL1 promoter) expressing the
LexA/B42 transcription factor or containing the empty vector were
used to detect compounds that directly inhibit the lacZ reporter
gene or that alter .beta.-galactosidase activity. The plasmids
employed were p424-GAL1 and p424-GAL1-TA (transcriptional
activator) with transformed cells selected on tryptophan-deficient
plates. Yeast expressing Caspase-1 activated by expression of high
levels of Asc (.DELTA.TEF3-Caspase-1-FLAG/TEF-HA-Asc) were used to
detect compounds that cross-react with hits from the
Fas/FADD/Caspase-8 screen. The Asc/Caspase-1 yeast cells were
cultured and assayed under identical conditions to those for the
primary NLRP1 HTS assay (see above), using cells expressing
6op-LEU2/2op-lacZ reporter genes and TEFFas-d-S1-TA substrate.
Cloning Systems for Caspase Activators.
[0139] To clone cDNAs encoding proteins that activate Caspase-1,
the yeast strain EGY191 containing 2op-LEU2/2op-lacZ was
transformed with plasmids containing transcriptional units of
TEF-Fas-d-S1-TA (substrate) and .DELTA.TEF3-Caspase1-FLAG
(pro-Caspase-1), as illustrated in FIG. 30B, and selected on
histidine-deficient plates. As controls, cells were subsequently
transformed with the Asc-expressing plasmid p424-TEF-HAAsc or empty
vector and selected on tryptophan-deficient plates.
[0140] To clone cDNAs encoding proteins that activate Caspase-2,
the yeast strain EGY48 containing 6op-LEU2/2op-lacZ was transformed
with plasmids containing transcriptional units of
.DELTA.TEF2-Fs-d-S2-TA (substrate) and .DELTA.GPD1-HA-Caspase2-FLAG
(pro-Caspase-2) and selected on histidine-deficient plates. As
controls, cells were subsequently transformed with the
RAIDD-expressing plasmid p424-TEF-HA-RAIDD or empty vector and
selected an tryptophan-deficient plates.
[0141] To clone cDNAs encoding proteins that activate Caspase-3,
the yeast strain EGY191 containing 2op-LEU2/2op-lacZ was
transformed with plasmids containing transcriptional units for
.DELTA.TEF3-Fas-d-S3-TA and .DELTA.CYC2-Caspase-3 and selected on
histidine-deficient plates. As controls, cells were transformed
with the Caspase-9-expressing plasmid p424-ADH-HA-Caspase9-FLAG or
empty vector and selected on tryptophan-deficient plates.
[0142] To clone cDNAs encoding proteins that activate Caspase-4,
the yeast strain EGY48 containing 6op-LEU2/2op-lacZ was transformed
with plasmids containing transcriptional units for GPD-Fas-d-S1-TA
and .DELTA.TEF2-Caspase4. As controls, cells were transformed with
a plasmid expressing high levels of (Caspase-7 (p424-ADHCaspase-7),
which results in active Caspase-7 did but does not cut the S1
site.
[0143] To clone molecules that activate Caspase-7, the yeast strain
EGY48 expressing 6op-LEU2/2op-lacZ/TEF-Fas-d-S3-TA/CYC1-Caspase-7
was created. The small amount of Caspase-7 exists as inactive
zymogen, and does not cut the S3 site. As a positive control,
pro-Caspase-7 was activated by expressing Caspase-9 at high levels
(p424-ADH-HA-Caspase9-FLAG). Active Caspase-9 did not cut the S3
site.
[0144] To clone cDNAs encoding proteins that activate Caspase-8,
the yeast strain EGY48 containing 6op-LEU2/2op-lacZ was transformed
with plasmids containing transcriptional units for GPD-Fas-d-S8-TA
and CYC1-Caspase8-HA and selected on histidine-deficient plates. As
controls, cells were transformed with FADD-expression plasmid
p424-TEF-HA-FADD or empty vector and selected on
tryptophan-deficient plates.
[0145] To clone cDNAs that activate Caspase-9, the yeast strain
EGY48 containing 6op-LEU2/2op-lacZ was transformed with plasmids
containing transcriptional units for TEF-Fas-d-S9-TA and
TEF-HA-Caspase9, on a low-copy plasmid p413 (CENLARS). The small
amount of pro-Caspase-9 exists as inactive zymogen, and does not
cut the S9 site. As controls, cells were transformed with the
Apaf-1*-expressing plasmid p424-TEF-HA-Apaf* or empty vector and
selected on tryptophan-deficient plates.
[0146] To clone cDNAs encoding proteins that activate Caspase-10,
the yeast strain EGY48 containing 6op-LEU212op-lacZ was transformed
with plasmids containing transcriptional units for GPD-Fas-d-S8-TA
and ADH-Caspase10-FLAG and selected on histiodine-deficient plates.
As controls, cells were transformed with the FADD expressing
plasmid p424-TEF-HA-FADD or empty vector and selected on tryptophan
deficient plates.
Procedures for cDNA Library Screening.
[0147] Screens for cDNAs encoding proteases (Caspases). To screen
for cDNAs encoding proteases capable of cleaving the S1 site
(WEHD), the yeast strain EGY48 containing 6op-LEU212op-lacZ was
transformed with p413-TEF-Fas-d-S1-TA followed selection on
histiodine deficient plates, then these cells were subsequently
transformed with a HEK293 cDNA library (contains TRP1 marker) or a
human placenta cDNA library (contains TRYP1 marker). The
transformants were seeded on tryptophan-deficient growth plates (SD
medium containing 2% glucose and 50 ug/ml leucine). Independent
colonies of 5.1.times.105 appeared from the HEK293 cDNA library in
48 hours. The colonies were harvested and pooled, and a portion of
the recovered cells (3.6.times.106 cells) was seeded onto
leucine-deficient selection plates (SD medium containing 1%
galactose, 0.2% raffinose, BU salts, and 80 ug/ml X-gal).
Bluecolored colonies appeared within a week and were subjected to
plasmid DNA extraction. The extracted plasmid DNAs were introduced
into KC8 E. coli cells by electroporation to efficiently recover
the cDNAs plasmids. The candidate cDNAs were again introduced into
the yeast cells containing 6op-LEU2/2op-lacZ/TEF-Fas-d-S1-TA or
6op-LEU2/2op-lacZ/TEF-Fas-d-G1-TA to confirm whether they cleave S1
specifically.
[0148] To screen for cDNAs encoding proteases capable of cleaving
the S3 site (DEVD), the yeast strain EGY48 containing
6op-LEU2/2op-lacZ, was transformed with p413-TEF-Fas-d-S3-TA,
followed by selection on histiodine-deficient plates, and
subsequently transformed with a HEK293 cell cDNA library, then
processed as above.
Screening for cDNAs Encoding Proteins That Activate Caspase-8 in a
FADD-dependent Manner.
[0149] The yeast strain EGY48 (6op-LEU2) was transformed with
plasmids p426-2op-lacZ/.DELTA.ADH1-FADD and
p413-TEF-Fas-d-S8-TA/CYC1-Caspase8, then subsequently transformed
with a HepG2 cDNA library, a human liver cDNA library, or a HEK293
cDNA library. The transformants were seeded on growth plates (SD
medium containing 2% glucose and 50 ug/ml leucine). Independent
colonies of 1.7.times.106 appeared from the HepG2 library in 48
hours. They were harvested, and pooled, and a portion of the cells
(2.4.times.107 cells) was seeded on selection plates (SD medium
containing 1% galactose, 0.2% raffinose, BU salts, and 80 ug/ml
X-gal). Blue-colored colonies appeared within a week, were
subjected to plasmid DNA extraction. The extracted plasmid DNAs
were introduced into KC8 E. coli cells by electroporation to
recover the cDNAs plasmids. The candidate cDNAs were again
introduced into the yeast cells containing 6op-LEU2/2op-lacZ and
plasmids containing transcriptional units for .DELTA.ADH1-FADD,
TEF-Fas-d-S8-TA, and CYC1-Caspase8; or TEF-Fas-d-S8-TA and
CYC1-Caspase8; or TEF-Fas-d-S8-TA to confirm whether they activate
Caspase-8 in a FADD-dependent manner.
Screening for cDNA Encoding Adapter Proteins That Link Fas or DR5
to Caspases-8 and -10.
[0150] The yeast strain EGY48 (6op-LEU2) was transformed with
p426-2op-lacZ/ADHDR5-FLAG and p413-TEF-Fas-d-S8-TA/CYC1-Caspase8,
then subsequently transformed with a HeLa cell cDNA library or a
HEK293 cDNA library. The transformants were seeded on growth plates
(SD medium containing 2% glucose and 50 ug/ml leucine). Independent
colonies of 1.0.times.106 appeared from the HeLa cell cDNA library
within 48 hours. They were harvested, pooled, and a portion of the
cells (2.4.times.107 cells) was seeded on selection plates (SD
medium containing 1% galactose, 0.2% raffinose, BU salts, and 80
ug/ml X-gal). Blue-colored colonies appeared within a week and were
subjected to plasmid DNA extraction. The extracted plasmid DNAs
were introduced into KC8 E. coli cells by electroporation to
recover the cDNA plasmids. The candidate cDNAs were again
introduced into the yeast cells expressing
6op-LEU2/2oplacZ/ADH-DR5-FLAG/TEF-Fas-d-S8-TA/CYC1-Caspase8,
6op-LEU2/2op-lacZ/TEFFas-d-S8-TA/CYC1-Caspase8, or
6op-LEU2/2op-lacZ/TEF-Fas-d-S8-TA to confirm whether they activate
Caspase-8 in a DR5-dependent manner.
[0151] The yeast strain EGY48 (6op-LEU2) was transformed with
p426-2op-lacZ/ADH-Fas and p413-GPD-Fas-d-S8-TA/CYC1-Caspase10, then
subsequently transformed with a HEK293 cell cDNA library. The
transformants were seeded on growth plates (SD medium containing 2%
glucose and 50 ug/ml leucine). Independent colonies of
2.2.times.106 appeared within 48 hours, and were harvested, and
pooled. A portion (3.2.times.107 cells) of the cells seeded on
selection plates (SD medium containing 1% galactose, 0.2%
raffinose, BU salts, and 80 ug/ml X-gal). Blue-colored colonies
appeared in a week and were subjected to plasmid DNA extraction.
The extracted plasmid DNAs were introduced into KC8 E. coli cells
by electroporation to recover the cDNA plasmids. The candidate
cDNAs were again introduced into the yeast cells expressing
6op-LEU2/2oplacZ/ADH-Fas/GPD-Fas-d-S8-TA/CYC1-Caspase10,
6op-LEU2/2op-lacZ/GPD-Fas-d-S8-TA/CYC1-Caspase10, or
6op-LEU2/2op-lacZ/GPD-Fas-d-S8-TA to confirm whether they activate
Caspase-10 in a Fas-dependent manner.
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[0209] All references cited herein are hereby incorporated by
reference in their entireties, whether previously specifically
incorporated or not. As used herein, the terms "a", "an", and "any"
are each intended to include both the singular and plural
forms.
[0210] Having now fully described the disclosed subject matter, it
will be appreciated by those skilled in the art that the same can
be performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the disclosure and without undue experimentation.
While this disclosure has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the subject matter
following, in general, the principles of the disclosure and
including such departures from the disclosure as come within known
or customary practice within the art to which the subject matter
pertains and as may be applied to the essential features
hereinbefore set forth.
[0211] Although the invention has been described with reference to
the above example, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
* * * * *